It is 1941 in San Francisco, and private investigators Sam Spade and Miles Archer are meeting with a prospective new client, Ruth Wonderly. Ruth appears to be very concerned for her sister and claims that she is missing. Explaining that she has been searching for her, with no success, Ruth wants to hire the private investigative duo to find her. Ruth's sister has been in a relationship with a man named Floyd Thursby and after she pays a very substantial retainer Archer agrees to follow her that night when she has an arrangement to meet with Thursby, to try to find her sister and bring her home.
In the middle of the night, a telephone call wakes Spade and he is informed that Archer has been murdered. He meets Detective Tom Polhaus, an old friend, at the murder scene, where surprisingly Polhaus and his partner Lieutenant Dundy have a multitude of questions for him about the new case, and specifically the woman to hired them. They then tell him that Thursby was also murdered during the evening, Dundy suggesting that Spade is the perpetrator, having both opportunity, and motive, to do so as it is probable that Thursby killed Archer and Dundy suspects Spade retaliated by killing Thursby.
Later that day, Spade meets with Ruth Wonderly whom he was unable to reach at her hotel the previous night. She is now going by the name Brigid O'Shaughnessy, and admits that although Thursby was her partner she does not know who killed him; she also admits he probably myrdered Archer. Although he find her story unconvincing, Spade agrees to investigate both murders.
Spade returns to his office and meets a shady character called Joel Cairo. Initially Cairo offers him five thousand dollars to find a black figure of a bird but subsequently pulls out a gun and demands to search the office. Spade manages to overpower Cairo, knocking him out and searching his belongings. When Cairo comes around he hires Spade. That evening, Cairo visits the office whilst O'Shaughnessy is there and it is immediately obvious that the two know each other already. When O'Shaughnessy mentions that the "Fat Man" is in San Francisco Cairo is visibly agitated.
The following morning, Spade pays a visit to Cairo's hotel and spots a young man by the name of Wilmer, whom he had already noticed following him earlier. He gives Wilmer a message for his boss, the Fat Man, Kasper Gutman. When Spade and Gutman meet, Gutman talks about the Maltese Falcon but his evasiveness angers Spade who leaves. Gutman in turn is angered by this, and sends Wilmer to bring him back at gunpoint.Although Spade easily overpowers Wilmer he sees Gutman regardless and this time Gutman tells the entire history of the Falcon. He offers Spade twenty-five thousand dollars plus a percentage of the sale, to find the bird.
Spade awakens in Gutmans suite finding himself alone and realizes his drink was spiked. He searches the suite and finds a newspaper with the arrival time of a freighter called La Paloma circled in it. He heads to the dock but by the time he arrives there the freighter is on fire and the ship's captain has been shot multiple times. He staggers into Spade's office clutching a parcel; he dies before Spade's eyes. The parcel contains the Maltese Falcon.
Spade answers the telephone to hear O'Shaughnessy's voice giving an address; he hears her scream then the line goes dead. Spade hides the parcel at the bus terminal then goes to the address O'Shaughnessy told him but it turns out to be an empty lot. When he returns to his home he realizes the purpose of her call was to get him away from his home so that she, Gutman, Cairo and Wilmer have time to break in and lie in wait for him. They draw their guns and Gutman gives Spade ten thousand dollars for the Falcon; Spade does not accept the money as part of his asking price is the person who murdered Archer, Thursby and Captain Jacobi. He believes this to be Wilmer. After dawn Spade asks his secretary to bring the Falcon but after inspection, Gutman declares the statuette a fake. During the ensuing exchange Wilmer flees. Gutman calms down, offering Cairo the chance to return to Istanbul with him to continue searching for the statuette. When they leave, Spade calls the police and tells them where they can find the two men, then turns his attention to O'Shaughnessy whom he knows to have murdered Archer in an attempt to implicate Thursby, her unwanted partner in crime. Although he has strong feelings for her, Spade turns her over to the police, neatly tying up all the loose ends of the case.