The novel opens on a diverse cast of characters in eighteenth-century Madrid. Ambrosio is a famous monk; Lorenzo is a wealthy young nobleman; and Antonia is a beautiful young woman. Lorenzo and Antonia meet while listening to Ambrosio, and fall in love instantly. Antonia's mother Elvira has come to the city seeking the assistance of Raymond, the Marquis de las Cisternas - one of Lorenzo's best friends.
Ambrosio is the most virtuous and strict monk in the whole city; when he discovers that a young nun named Agnes has gotten pregnant despite her vows, he turns her over to the cruel Prioress with no regrets. His lack of sympathy comes to haunt him, however, when a young monk named Rosario is revealed to be the beautiful Matilda, a woman who has come to the monastery out of love for Ambrosio. The monk struggles with his desires, but eventually succumbs to temptation and breaks his vows of chastity by having sex with Matilda.
Lorenzo is stunned to discover that Raymond is the lover of Agnes, Lorenzo's sister and a nun. Raymond explains that he and Agnes fell in love over the course of Raymond's dramatic journey through Europe, an odyssey that involved sly bandits, vengeful aunts, and frightening ghosts. Agnes's parents vowed her to a convent, but she and Raymond met nightly to declare their love for each other. Raymond impregnated Agnes during one of these nightly visits, and now he vows that he will rescue her and make her his wife. Lorenzo decides to assist his friend and his sister, and the two men attempt to rescue Agnes from the convent. However, the Prioress tells the men that Agnes has died from a mysterious illness.
Lorenzo visits Elvira, Antonia's mother, to ask for her daughter's hand in marriage. However, Elvira is a common woman who married a Marquis, and she knows that there are numerous problems inherent in lowborn women marrying noblemen. Still, Lorenzo vows to help Elvira and Antonia.
Meanwhile, Ambrosio sees Antonia in church and immediately begins to desire her. He makes frequent visits to her family's house under the pretense of offering counseling to the gravely ill Elvira, but in truth he wants to get closer to Antonia. One day, Ambrosio molests Antonia, but the watchful Elvira drives him away before he can cause any major harm to the girl.
Ambrosio despairs of being able to possess Antonia. Matilda, however, offers her assistance, summoning a demon that gives Ambrosio a magical myrtle branch. The myrtle unlocks the doors to Antonia's house and puts her in a deep sleep. Just as Ambrosio is about to violate her, Elvira appears. In the confrontation that ensues, Ambrosio kills Elvira and flees.
Raymond has fallen into a deep depression following news of Agnes' death. However, his faithful servant Theodore disguises himself as a beggar, and receives a message from one of the nuns of St. Clare's convent. The message states that the men need to arrest the Prioress if they want to get revenge on Agnes' murderers. Upon seeing the note, Lorenzo springs into action to avenge his sister.
Antonia is wild with grief for her mother. One lonely night, she claims to see her mother's ghost and faints in terror. The landlady, Jacintha, runs to the monastery and calls upon the most revered monk in Madrid - Ambrosio. Upon Matilda's advice, Ambrosio brings with him a drug that will induce a sleep as heavy as death, and gives it to Antonia. When the doctor declares Antonia dead, Ambrosio takes her body to the crypt of the monastery, knowing that she will awake in a few hours and she will be all his.
At the procession of St. Clare, Lorenzo and his men arrest the Prioress. The Mother St. Ursula, who left the note for Theodore, publicly explains how the Prioress tormented Agnes and poisoned her to death. The crowd is so infuriated that they kill the Prioress and sack the convent of St. Clare's. Lorenzo is protecting a group of nuns in the crypt when he finds a secret staircase. At the bottom is Agnes, confined for months with only a little bread and water. Lorenzo brings his sister to safety.
Meanwhile, Ambrosio rapes Antonia in the dark crypt. Weeping, Antonia attempts to escape, but Ambrosio follows her and stabs her to death to keep her from escaping. Antonia dies in Lorenzo's arms.
Raymond and Agnes are reunited, and marry. Lorenzo mourns Antonia, but eventually marries the beautiful Virginia de Villa-Franca. Ambrosio and Matilda are handed over to the Inquisition, and accused of rape, murder, and sorcery. Ambrosio is tortured in an attempt to make him confess. At Matilda's urging, Ambrosio eventually signs his soul over to the devil in order to avoid execution. However, the devil reveals a number of shocking facts: Elvira was Ambrosio's mother and Antonia was his sister, and Matilda was just a demon who took on human form. Chiding Ambrosio for asking only for release from prison rather than long life or wealth, the devil leaves Ambrosio to eagles that rip him apart and drop him on sharp rocks. The monk dies in agony, and his body washes into a river.