Sister Aloysius Beauvier is a nun and principal at a Catholic school in 1964 New York. Her strict methods put her at odds with Father Flynn (Philip Seymour Hoffman), the new, more liberal priest and, after a sermon about doubt, Sister Aloysius asks the other nuns to be on the look out for anything unusual that may have prompted the subject matter.
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Sister James (Amy Adams) begins to notice Father Flynn and Donald Miller, the school's only black child. One day, Donald is called to the rectory and, when he returns, seems sullen. Sister James also notices the smell of alcohol on his breath. She later sees Father Flynn placing a shirt inside Donald's locker.
With nowhere else to turn, she reports this to Sister Aloysius, who confronts Flynn in his office. Father Flynn tells the women to leave it be, but after pressing, tells them Donald had drank alter wine and that he was hiding it to protect the boy. The following Sunday, Father Flynn delivers a sermon on gossip.
Sister James is relieved, but Aloysius is undaunted, - sure of what has occurred. She presses further, contacting Donald's mother, who tells her Donald's father is abusive and that he needs to continue attending the school to go to a decent high school.
Aloysius again confronts Father Flynn, who maintains nothing occurred. But Aloysius is certain, leading to a standoff and a threat to involve the Bishop. Will the Father succumb to her demands? The answer reveals as much about her as it does Father Flynn - and the nature of the doubt we all harbor.