The police arrest Maddalena Anna Paradine (Alida Valli) for poisoning her late husband, Col. Paradine. She claims innocence, and hires solicitor Sir Simon Flaquer (Charles Coburn) and barrister Anthony (a.k.a Tony) Keane (Gregory Peck, with an occasional British accent) as her defense team. Anthony Keane is very successful and happy at both his career and in his marriage with Gay (Anne Todd). Sir Flaquer and Keane have been friends for many years, and Gay considers Judy Flaquer (Joan Tetzel) her best friend. The presiding judge, Lord Thomas Horfield (Charles Laughton) and his wife, Lady Sophie (Ethel Barrymore) represent the legal and popular views about the case.
During the pretrial period, Keane's affections changes, as he falls under Maddalena's spell, starting at their first meeting. Gay Keane realizes her husband has fallen for Mrs. Paradine. She confides in Judy, who advises her to confront Keane about this. But Gay refuses to leave or condemn him She knows he still loves and values her, because Keane has not chosen to leave, yet. In fact, Gay wants Mrs. Paradine to be acquitted so she can fight against a living woman instead of a lost love.
The suspects narrow down to Maddalena or Andre LaTour (Louis Jourdan), Col Paradine's valet of many years. She claims to love her blind husband. Their marriage had no problems. Andre argued with Col. Paradine earlier that night, about an unknown subject, and was dismissed. There is great uncertainty in the motives and actions of many people that fateful evening. The butler brought Col Paradine that glass of brandy, something he rarely requested at bedtime. Maddalena claims to have laid out her husband's sleepwear but did not go near the brandy. Andre was pounding on Col Paradine's bedroom door, and saw Maddalena enter her room after he started. Andre said the brandy glass still had a few drops when he forced his way inside. However, the police found the brandy glass cleaned and wiped. Just before the trial starts, Andre warns Keane that Maddalena is evil and he hates her because she brought so much turmoil into their lives.
As a defense witness, Andre claims that Col. Paradine dismissed him as his valet, but to be reassigned to some other household role. He did put down the Colonel's hunting dog last year, but claimed to have disposed of the poison at that time. The glass still contained some brandy when he went to find the butler and call the doctor. Under more pressure, Andre reveals that he was Maddalena's lover and had confessed to Col. Paradine that day. At this point,Maddalena forces Keane to stop this line of questions. Disillusioned, Keane realizes that she has hidden motives for both her husband's murder and not implicating Andre as the murderer. The next day, Keane puts her on the stand and proceeds to question her relationship with Andre. Shortly after this begins, Sir Flaquer tells Keane of Andre's suicide. At this news, Maddalena breaks down and confesses to poisoning her husband out of love for Andre, who had come to hate her for betraying his Colonel. She receives the death sentence, and Keane goes back to his wife, sadder but more appreciative of his wife's love.