The play is set in the fictional St. Nicholas Church School, in the Bronx, during the fall of 1964. It opens with a sermon by Father Flynn, a beloved and progressive parish priest, addressing the importance of uncertainty: "Doubt can be a bond as powerful and sustaining as certainty." The school's principal, Sister Aloysius, a rigidly conservative nun who is vowed to her order, the Sisters of Charity, insists upon constant vigilance. During a meeting with a younger nun, Sister James, Aloysius reveals a deep mistrust toward her students, her fellow teachers, and society in general. Naïve and impressionable, James is easily upset by Aloysius's severe manner and harsh criticism.
Aloysius and Flynn are put into direct conflict when she learns from James that the priest had a one-to-one meeting with Donald Muller, St. Nicholas's first African-American student. Mysterious circumstances lead her to believe that sexual misconduct occurred. In a private meeting, purportedly regarding the Christmas pageant, Aloysius, in the presence of James, openly confronts Flynn with her suspicions. He angrily denies wrongdoing by insisting that he was disciplining Donald for drinking altar wine and claims to have been protecting the boy from harsher punishment. James is relieved by the explanation. Flynn's next sermon is on the evils of gossip.
Aloysius, dissatisfied with Flynn's story, meets with Donald's mother, Mrs. Muller. Despite Aloysius's attempts to shock her, Mrs. Muller says she supports her son's relationship with Flynn. She ignores Aloysius's accusations. Before departing, she hints that Donald may be "that way," which may cause her husband to be beating him.
Flynn eventually threatens to remove Aloysius from her position if she does not back down. Aloysius informs him that she phoned the last parish to which he was assigned and that she discovered a history of past infringements. After declaring his innocence, the priest begins to plead with her, but she blackmails him and demands that he resign immediately, or she will publicly disgrace him with his history. Disgusted, she leaves the office. Flynn calls the bishop to apply for a transfer, and is subsequently promoted to pastor of a nearby parochial school.
After hearing the news, Aloysius reveals to Sister James that the decisive phone call to Flynn's previous parish was a fabrication and that she has no evidence of past wrongdoing. As a result, Aloysius is left with ambiguous doubt, and the audience is left to wonder if the doubt is in either herself or the Church. With no proof of Father Flynn's guilt or innocence, the audience is left with its own doubt.
