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," he says). The school's principal, Sister Aloysius, a rigidly conservative nun vowed to the order of 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.

Sister Aloysius and Father Flynn are put into direct conflict when she learns from Sister James that the priest met one-to-one 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 Sister James, openly confronts Flynn with her suspicions. He angrily denies wrongdoing, insisting that he was disciplining Donald for drinking altar wine, claiming to have been protecting the boy from harsher punishment. James is relieved by his 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", and that her husband may be beating him because of this.

Father Flynn eventually threatens to remove Aloysius from her position if she does not back down. Aloysius informs him that she previously phoned the last parish he was assigned to, discovering a history of past infringements. After declaring his innocence, the priest begins to plead with her, at which point she blackmails him and demands that he resign immediately, or else she will publicly disgrace him with his history. She leaves the office, disgusted. Flynn calls the bishop to apply for a transfer, where, later, he receives a promotion and is instated as pastor of a nearby parochial school.

Learning this, Aloysius reveals to Sister James that the decisive phone call to Flynn's previous parish was a fabrication and 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 that Father Flynn is or is not innocent, the audience is left with its own doubt.

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