7 Khoon Maaf tells the story of an Anglo-Indian woman, Susanna Anna-Marie Johannes (Priyanka Chopra), who murders all her husbands. Susanna tries to find love, but six of her seven husbands have a flaw which proves fatal. The killings (and her yearning for love) are explained by the loss of her mother at a young age. The story is told by a young forensic pathologist, Arun, (Vivaan Shah) to his wife (Konkona Sen Sharma). Arun has watched Susanna since their childhood, and has a secret crush on her. He owes his life to her; she funded his education, making him what he is today. Arun tells his wife that Susanna has committed suicide, leaving him a note congratulating him on his marriage. The doctor must now confirm that the body found is Susanna's. Arun is unable to overcome his grief over her death and pours out his heart to his wife, who listens patiently.
Susanna's first husband—Edwin Rodriques (Neil Nitin Mukesh), an army major—is overbearing, jealous and possessive. Disabled, he cannot believe that a beautiful woman like Susanna would be faithful to him. Uncertain of his sexual potency, he takes out his anger on his wife. Although Susanna endures his mental cruelty, she cannot forgive Edwin for blinding her faithful, mute stableboy (Shashi Malviya) with a whip. Edwin is eliminated during a panther-hunting trip with the help of Susanna's faithful maid (Usha Uthup), butler (Harish Khanna) and the mute stableboy.
Susanna's second husband—Jamshed Singh Rathod (John Abraham), who renames himself Jimmy Stetson after their marriage—is a singer whose flaw is pride. The marriage begins well, but Jimmy becomes successful and misuses his new-found fame. He steals songs, dallies with other women and becomes dependent on drugs. Susanna wants to be lucky in love the second time; she tries to wean him from his addiction, but he continues in secret. She gives up, disposing of him with a heroin overdose. Police investigators find footprints near his body indicating that a person with six toes is the murderer.
Susanna's third husband—Wasiullah Khan (also known as Musafir) (Irrfan Khan)—is a soft-spoken, thoughtful poet by day and a sado-masochist by night. Susanna tries to cover her bruises with makeup; her servants cannot bear to see her mistreated, and advise her to get rid of him. Khan is ultimately interred in a snowy Kashmiri grave.
Her fourth husband, Nicolai Vronsky (Aleksandr Dyachenko), is a Russian spy leading a double life. When Susanna discovers he has another wife and family, her rage knows no bounds and he meets his end with the aid of Susanna's pet snakes. After Vronsky's death, Susanna no longer agonizes over eliminating anyone of whom she disapproves.
Her fifth husband, Keemat Lal (Annu Kapoor), is a police inspector who has shielded his "Madame" from prosecution for two murders in exchange for marriage. With a voracious appetite for sex, his dependence on Viagra proves his undoing; one fateful night, Susanna mixes an overdose of the drug into Lal's drink.
Modhusudhon Tarafdar (Naseeruddin Shah) is a Bengali doctor who rescues Susanna from a suicide attempt (when she learns about Arun's marriage) and puts her on a mushroom-only diet. Although she does not want to marry him, Tarafdar persuades her with a promise that she will be the sole heir of his property. Actually bankrupt, he tries to poison Susanna with mushroom soup several years later for her inheritance. Her butler drinks it by accident and dies instead; in a state of shock, she shoots Tarafdar during a game of Russian roulette. That night, Susanna sets her house ablaze in another suicide attempt. Seeing the house on fire, her maid (who has six toes) tries to rescue her but dies in the attempt. In the forensics laboratory, Arun discovers that the body is not Susanna's; however, he declares Susanna dead in his reports. He looks for her; when he finds Susanna, she tells him she is finally getting married the next day to someone who accepts her, knowing all her sins. In an epiphany she imagines Jesus Christ as her seventh husband, full of love and never hurtful; she dies, becoming a nun and finding the love she sought all her life. Arun and his wife return home after he tells her that Susanna is dead.