Note: the entire movie is filmed in real time with a single camera like a one-act stage play to appear like one continuous seamless shot (one-take).
Two brilliant young aesthetes, Brandon Shaw (John Dall) and Phillip Morgan (Farley Granger), strangle to death their former classmate from Harvard University, David Kentley (Dick Hogan), in their apartment. They commit the crime as an intellectual exercise; they want to prove their superiority by committing the "perfect murder". Phillip is the one who strangles David with a piece of rope while Brandon restrains David until he dies.
After hiding the body in a large antique wooden chest, Brandon and Phillip prepare to host a dinner party at the apartment, which has a panoramic view of Manhattan's skyline. Brandon decides to use the chest containing the body as a buffet table for the food, just before their housekeeper, Mrs. Wilson (Edith Evanson) arrives to help with the party. "Now the fun begins," Brandon says when the first guests arrive.
The guests, who are unaware of what has happened, include the victim's fiancée, Janet Walker (Joan Chandler) and her former lover Kenneth Lawrence (Douglas Dick), who was once David's close friend. Also arriving is the victim's father Mr. Kentley (Cedric Hardwicke) and chatty aunt Mrs. Atwater (Constance Collier); his mother is not able to attend due to a cold.
Brandon and Phillip's idea for the murder was inspired years earlier by conversations with their prep school housemaster, publisher Rupert Cadell (James Stewart). While at school, Rupert had discussed with them, in an apparently approving way, the intellectual concepts of Nietzsche's Übermensch, and De Quincey's art of murder, as a means of showing one's superiority over others. He too is among the guests at the party and the last one to arrive, since Brandon in particular feels that he would approve of their "work of art".
Brandon's subtle hints about David's absence indirectly lead to a discussion on the "art of murder". The self-assured Brandon appears calm and in control, although when he first speaks to Rupert he is nervously excited and stammering. Phillip, on the other hand, is visibly upset and morose; aware that Rupert is the only person who might suspect what they just did. Phillip does not conceal it well and starts to drink too much.
As the evening goes on, David's father and fiancée begin to worry that he has neither arrived nor phoned. Brandon increases the tension by playing matchmaker between Janet and Kenneth. Mrs. Kentley calls, overwrought because she has not heard from David, and Mr. Kentley decides to leave, as does Janet and Kenneth. Mr. Kentley takes with him some books Brandon has given him, tied together with the same rope Brandon and Phillip used to strangle his son.
However, Rupert returns to the apartment a short while after everyone else has departed, pretending that he has left his cigarette case behind. He hides the case behind some books on the chest, asks for a drink and then stays to theorize about David's disappearance. He is encouraged by Brandon, who hopes Rupert will understand and even applaud them. A drunk Phillip is unable to take it any more; he throws a glass and says, "Cat and mouse, cat and mouse. But which is the cat and which is the mouse?"
Now, Rupert lifts the lid of the chest and finds the body inside. He is horrified but also deeply ashamed, realizing that Brandon and Phillip used his own rhetoric to rationalize murder. Rupert now disavows all his previous talk of superiority and inferiority, realizing that there is no way to objectively define these concepts, then seizes Brandon's gun and fires several shots out the window in order to attract attention. As approaching police sirens get louder, Rupert pulls up a chair next to the chest, while Brandon casually pours himself a drink, and the distraught Phillip notes that the police are coming and begins to play the piano a final time as the image fades out and the film's end credits appear on the screen.