Newly arrived in Madrid, Leonella and her niece Antonia visit a church to hear the sermon of a celebrated priest, Ambrosio, and while waiting tell their story to two young men, Don Lorenzo and Don Christoval. Antonia's Grandfather is the Marquis de las Cisternas, who was unhappy with his son’s marriage, causing her parents to flee, leaving their young son behind only to be told a month later he has died. Leonella has come to Madrid to convince the Marquis’ son, Raymond de las Cisternas, to resume their pension, which has been cut off. As the story is told, Lorenzo falls in love with Antonia. The mysterious priest, who was left at the abbey as a child, delivers the sermon, and Antonia is fascinated with him. Lorenzo vows to win the hand of Antonia, but must first visit his sister Agnes, who is a nun at the nearby abbey. Having fallen asleep in the church, he awakens to find someone delivering a letter for his sister from Raymond de las Cisternas. On the way home, a gypsy warns Antonia that she is about to die, killed by someone who appears to be honorable.
Ambrosio is visited by nuns, including Agnes, for confession. She drops a letter which reveals her plans to run away with Raymond de las Cisternas. When Agnes confesses that she is pregnant with Raymond’s child, Ambrosio turns her over to the prioress of her abbey for punishment. As she is led away, she curses Ambrosio. Returning to the abbey, Ambrosio's constant companion, a novice named Rosario admits that he is a woman named Matilda, who disguised herself so that she could be near Ambrosio. They both know he must throw her out of the monastery, but she begs him not to, and vows to kill herself if he does. He relents, but after talking the next day she decides to leave of her own accord, on the condition Ambrosio gives her a rose to remember him by. As he picks the rose, he is bitten by a serpent and is rushed to his room where it is predicted that he will die within three days. Rosario acts as his nurse, and the next day it is discovered that Ambrosio is cured which is proclaimed a miracle. When the other monks leave, Matilda reveals that she sucked the poison from Ambrosio’s wound and is now dying herself. At the point of death, she begs him to make love to her, and he succumbs to the temptation at last, having discovered that she is the model who sat for his beloved portrait of the virgin Mary.
Lorenzo confronts Raymond about his relationship with his sister Agnes and his being identified as Alphonse d’Alvarada, who tried to elope with her. Raymond tells a story of the time he went travelling in Germany with his rank concealed under the name Alphonse d’Alvarada. While traveling, his chaise is incapacitated and His servant finds him some lodging at a nearby cottage owned by Baptiste and his wife, who is anything but congenial. Another party, a baroness and her retinue also stop for the night. Receiving a sign of bloody sheets on his bed from Marguerite, Baptiste’s wife, Alphonse realizes that something is amiss, and discovers that he has fallen into a group of murderers, who waylay travelers to kill and rob them. He avoids being drugged and manages to escape with the others, along with Marguerite, who kills Baptiste. They make it to Strasbourg, where Marguerite shares her story of illicit love with a bandit, by whom she has two children, and being forced into marriage with Baptiste. She returns to the home of her father, and Raymond continues his travels, taking along Marguerite’s son, Theodore, as a servant.
At the home of the baroness Raymond falls in love with her niece Agnes, and goes to the baroness to ask for her blessing. However The Baroness is in love with Raymond and when he refuses her advances since he loves Agnes, she vows vengeance. Discovering that it is Agnes, she plans to send her to the convent and so Raymond and Agnes make plans to elope. Agnes plans to dress as the bleeding nun, a ghost who haunts the castle, when she escapes with Raymond. The two drive away in the night, but the carriage crashes, and when Raymond awakens, he finds the nun Agnes is gone. After several months healing, he learns that it was not Agnes but the bleeding nun herself who was with him. Raymond learns that the bleeding nun is an ancestor, and he is responsible for burying her bones and so release her from her hauntings. He finds Agnes in the convent and takes the disguise of the convent gardener. There he overcomes Agnes, earning her rejection. However, when she discovers that she is pregnant, she begs him to come to rescue her.
When Raymond finishes his story, Lorenzo agrees to help him elope with Agnes. Lorenzo then goes to visit Elvira, who is Raymond’s sister-in-law and the mother of Antonia, to ask for permission to court Antonia. However, Elvira is very fearful that her daughter might be rejected by Lorenzo’s family, just as she was rejected by the Cisternas. Despite Lorenzo’s pleadings, Elvira suggests to both Raymond and to Antonia that they resist their love. Lorenzo promises that he will get his family’s blessing and marry Antonia. In the meantime, Lorenzo tries to visit his sister Agnes in the convent, but is told that she is too ill to see him. He has sent to Rome to receive a papal bull releasing Agnes from her vows so that she may honorably marry Raymond without fear of retribution. When the prioress of the abbey is presented with the papal bull, she tells Lorenzo that his sister died several days before. Lorenzo does not believe it, but knows that is simply the prioress’s way to relieve the shame that having a pregnant nun would have on the abbey. However, after two months, there is no other word concerning Agnes. In the meantime, he has secured his family’s blessing on his hoped-for marriage with Antonia.
Ambrosio and Matilda spend the night making love, Ambrosio no longer feeling the guilt of sin. The next night in the cemetery, she performs some ritual of which Ambrosio can only see flashes of light and quaking of the ground; when she returns, she is free of the poison, and free to be Ambrosio’s secret lover. But as the week progresses, Ambrosio grows tired of her, and his eyes begins to wander, noticing the attractiveness of other women. Ambrosio is approached by Antonia, who asks him to provide a confessor for Elvira, her dying mother, and is immediately attracted to her. He prays for Elvira, who begins to improve, and so agrees to come to visit them often, for the simple purpose of being with Antonia and hopefully seducing her. Elvira confesses that she sees something familiar in Ambrosio, but she cannot pinpoint what it is.
Ambrosio continues his visits to Antonia. He asks if there is not a man whom she has ever loved, and she confesses that she loves him. Misinterpreting her, he embraces her, but she resists him, insisting that she did not love him in that way, yet the priest continues to ravish her until her mother enters. Ambrosio pretends that nothing was happening, but Elvira had already suspected his designs on her daughter and tells him that his services are no longer needed. Matilda comes to his room and tells him she can help him to gain Antonia’s charms, even though she realizes she herself no longer holds his interest, in the same way in which she was healed of the poison: witchcraft. Ambrosio is horrified and rejects her suggestion. However, when she shows him a magic mirror that reveals to him Antonia bathing, he agrees. Matilda and Ambrosio return to the cemetery, where Matilda calls up Lucifer and receives his help, and they receive a magic myrtle bough, which will allow Ambrosio to open any door, as well as satisfy his lust on Antonia without her knowing who is her ravisher. Ambrosio agrees, without, he believes, selling himself to the devil.
Raymond mourns the death of his lover, Agnes, so Theodore plots to disguise himself as a beggar and go to the convent to find out what happened to her. He is taken into the convent, where he hopes that Agnes will recognize him, sending some word of her state. He is disappointed when no word comes. However, as he leaves, Mother St. Ursula, hands him a basket with gifts. Theodore takes the basket back to Raymond, where they find a note hidden in the linen cover, stating that they should have the cardinal arrest both Mother St. Ursula and the prioress, so that Agnes’s murder can be requited. Ambrosio carries out his plot to rape Antonia. With the magic myrtle bough he enters her chamber and finds her asleep. He performs the magic rite that will prevent her resistance. He is on the point of raping her when Elvira enters the room and confronts him, promising that she will make his true nature public. In desperation, Ambrosio murders Elvira without carrying out his true purpose of ravishing Antonia. He returns to the abbey, unsatisfied in his lust and horrified that he has now become a murderer.
Antonia is grief-stricken at the death of her mother and alone. Leonella is married and distant, Raymond is ill and ignorant of her plight, and Lorenzo has gone to get an arrest order for the death of his sister. One night Antonia wanders into Elvira’s room and sees what she takes to be her mother’s ghost, which warns her that it will return in three nights and Antonia will die. Terrified, Antonia faints and is found by her landlady, Jacintha, who goes to Ambrosio, requesting him to exorcise her home. Under Matilda’s advice, Ambrosio acquires a concoction that will induce a condition appearing to be death for Antonia. While he is attending Antonia, he slips the potion into her medicine and waits. While he is waiting, he sees what he fears is, in actuality, the ghost of Elvira retreat across the room. He pursues it and discovers it is Flora, Antonia’s maid, who is spying on him on the advice of Elvira before she died. As they are speaking, Jacintha cries out that Antonia is dying, as it indeed appears. With her "dying" breath, Antonia confesses how much she admired Ambrosio and desired his friendship, against her mother’s wishes. She leaves everything to her aunt Leonella, and releases her half-uncle Cisternas from all obligations to her, though she waited for him to come rescue her from her dire straits.
Lorenzo arrives back in Madrid with a representative of the Inquisition. During the procession honoring St. Clare, the prioress is arrested. Mother St. Ursula publicly relates the account of Agnes’s trial by the sisters. The majority voted for the most extreme punishment, which would entail Agnes being thrown in a dungeon and left for dead. However, at Mother St. Ursula’s instigation, the punishment is mitigated to death by poison. At Mother St. Ursula’s revelation that the prioress is a murderer, the crowd turns to rioting. Despite the Inquisitor’s pleas, she is attacked and killed by the crowd. Then they turn on the other nuns, vowing that all of them must be destroyed and the convent torn down. In the confusion, Lorenzo finds a group of nuns and a young woman named Virginia hiding in the cemetery vault near the statue of St. Clare. Groans coming from the statue arouse Lorenzo’s suspicions. He manages to move the statue to find a passage leading down into a dungeon, where he finds Agnes, alive and holding the body of her baby. Lorenzo removes Agnes from the dungeon and with Virginia’s help, takes the group of nuns to safety.
When Antonia awakens from her drugged sleep in the crypt Ambrosio rapes her. Afterwards, he is as disgusted with Antonia as he was with Matilda, who comes to warn him about the riot. Ambrosio kills Antonia in her attempt to escape. Virginia visits Lorenzo as he is recovering from his grief, and the two become closer. Lorenzo convinces Agnes to tell of her experiences at the hands of the prioress. She tells of having awakened to the horrors of the tomb. With the putrid conditions of her surroundings and the pangs of hunger not expected to be assuaged, she many times contemplates suicide, but the thought of her unborn child prevents her. At length she is visited by the prioress, who admits that she purposely gave her an opiate rather than poison, so that she could carry out the punishment that she sees as fitting for Agnes’s sin. She will be imprisoned in the dungeon, with enough food to ensure her survival, nothing more. In the dungeon, Agnes gives premature birth to her baby, which soon dies. At length, no food is brought, and Agnes resigns herself to die, when she is rescued by Lorenzo. Agnes and Raymond are married, and the couple leaves Madrid for Raymond’s castle, accompanied by Lorenzo and Virginia, who are also eventually married.
Ambrosio and Matilda are brought before the Inquisition, and at first both proclaim their innocence, but then Matilda confesses her guilt and is condemned to be burned at the auto-da-fé. Ambrosio insists upon his innocence and is tortured. When returned to his cell to regain his strength for a second "questioning", he is visited by a vision of Matilda, who tries to convince him to completely yield his soul to Satan as she has. She leaves him the volume by which the rite is performed. Ambrosio again proclaims his innocence, but when faced with the instruments of torture once again, he admits to his sins of rape, murder, and sorcery and he too is condemned to burn. In despair, Ambrosio requests Lucifer to save his life, who tells him it will be at the cost of his soul. Yet still Ambrosio resists, hoping eventually for God’s pardon. Lucifer informs him that there is none, and Ambrosio, after much resistance, signs the contract. He is rescued from the cell by Lucifer and brought to a wilderness. Lucifer informs him that Elvira was his mother, making Antonia his sister, adding to his crimes the sin of incest. Lucifer reveals that it has long been his plan to gain Ambrosio’s soul, and Matilda was his servant in the process. Lucifer then carries Ambrosio up and drops him on the rocks below. Ambrosio suffers for six days, dying alone and damned for eternity.