The setting is 1940s San Francisco. 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 she is missing. Explaining that she has been searching for her with no success, Ruth wants to hire the detective duo to find her. Ruth's sister has been in a relationship with a man named Floyd Thursby, and after Ruth pays a very substantial retainer, Archer agrees to follow her that night, when she has an arrangement to meet with Thursby, to try to retrieve her sister.
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. Later, upon returning home, Sam is visited by Tom and his partner Lieutenant Dundy, who grill him about his new case, before revealing that Floyd Thursby was also murdered that night. Dundy suggests that Spade is the perpetrator, having both opportunity, and motive; it is probable that Thursby killed Archer and Dundy suspects that Spade retaliated by killing Thursby.
Later that day, Sam 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 murdered Archer. Although he finds her story unconvincing, Sam agrees to investigate both murders for her.
Sam returns to his office, where he is visited by a shady character called Joel Cairo. Initially Cairo offers him $5000 to find a black statue of a bird, but subsequently pulls out a gun and demands to search the office. Sam manages to overpower Cairo, knocking him out and searching his belongings. When Cairo comes to, he hires Spade. That evening, Cairo visits Sam's apartment while Brigid is there and it is immediately obvious that the two know each other already, and have a contentious relationship. Brigid and Cairo allude to the fact that someone referred as the "Fat Man" is in San Francisco. Meanwhile, a boy in a trench coat, who has been following Sam all day, spies on the apartment from the street.
The following morning, Spade pays a visit to Cairo's hotel and spots the boy in the trench coat who has been following him, who goes by the name of Wilmer. He warns Wilmer to stop following him, and soon learns that the boy works for the infamous "Fat Man," Kasper Gutman. Gutman calls Sam's office and invites him to take a meeting. When Spade and Gutman meet later, Gutman tells him about the Maltese Falcon, a valuable statuette that he is searching for, but his evasiveness appears to frustrate Sam, who leaves abruptly. Gutman in turn sends Wilmer to retrieve Sam later that day. When Sam comes back, Gutman tells the entire history of the Falcon. He offers Sam $25,000 plus a percentage of the sale (likely 1/4 of $1,000,000) to find the bird.
Sam awakens in Gutman's suite finding himself alone and realizes that his drink was spiked. He searches the suite and finds a newspaper with the arrival time of a freighter called La Paloma circled in black pen. He heads to the dock, but by the time he arrives the freighter is on fire. Going back to his office, Sam tries to piece together the various events when he and his secretary are interrupted by the captain of the La Paloma, who has been shot multiple times, and staggers into Sam's office clutching a parcel. He dies on the spot, and Sam and Effie discover that the parcel contains the Maltese Falcon.
When the telephone rings, Effie answers it and speaks to Brigid, who gives an address; suddenly Brigid screams and the line goes dead. Hysterical, Effie sends Sam to find Brigid. Sam tells Effie to notify the police, but not to tell them about the Maltese Falcon. Sam hides the parcel with the falcon at the bus terminal in a safe deposit, then goes to the address Brigid gave, but it turns out to be an empty lot. When he returns to his home he realizes the purpose of Brigid's call was to get him away from his home so that she, Gutman, Cairo and Wilmer would have time to break into his apartment and wait for him there. As he enters his apartment to find them, Gutman offers Sam $10,000 for the Falcon. Sam tries to strike a bargain whereby they offer Wilmer to the police as a fall man and all run away from the crime unpunished. He also asks for answers about who really murdered Archer, Thursby and Captain Jacobi. After Gutman reveals some answers, Sam calls Effie and tells her to bring the Falcon. When it arrives, Gutman inspects it, before declaring it a fake. Wilmer flees. Gutman invites Cairo 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 men, then turns his attention to Brigid whom he now 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.
