In 1941, SS Colonel Hans Landa (Christoph Waltz) arrives at a dairy farm in France to interrogate Perrier LaPadite (Denis Menochet) about rumors that he is hiding a local Jewish family of dairy farmers that has disappeared. By leaving a subtle hint over promising potential immunity for LaPadite's family, Landa breaks LaPadite down and persuades him to confess to hiding the family underneath his floor. Landa then orders his men into the house to shoot the floorboards where they are hiding underne. Only the 18-year-old Shosanna (Mélanie Laurent) escapes.
In the spring of 1944, Lt. Aldo Raine (Brad Pitt) is tasked to recruit a team of eight Jewish-American soldiers to go behind German lines and strike fear in all enemy soldiers. He tells his team that they each owe him 100 Nazi scalps. They operate with a "take-no-prisoners" policy and come to be known among the ranks of the German Army as the "Basterds". One survivor of an attack, a Private named Butz (Sönke Möhring), is interviewed by Adolf Hitler (Martin Wuttke). Butz's account of the attack is shown in flashback: His squad was ambushed and his dead comrades were scalped. His squad leader was beaten to death upon refusing to reveal the location of a German position the Basterds intend to ambush, which Butz later agrees to. Butz then removes his cap and reveals the swastika Raine carved into his forehead.
In June 1944, Shosanna has assumed a new identity as "Emmanuelle Mimieux" and is operating a cinema in Paris. She meets Fredrick Zoller (Daniel Brühl), a German sniper whose exploits have been celebrated in a propaganda film, Stolz der Nation (Nation's Pride). Zoller is attracted to Shosanna and arranges a meeting with Joseph Goebbels (Sylvester Groth), and convinces Goebbels to hold the premiere at Shosanna's cinema. Shosanna realizes that the presence of several high-ranking Nazi officials provides an opportunity to avenge her family and resolves to burn down the cinema during the premiere, using hundreds of reels of highly combustible nitrate film.
At the same time, the British also learn of the premiere, and Winston Churchill (Rod Taylor) and General Ed Fenech (Mike Myers) dispatch former film critic Lt. Archie Hicox (Michael Fassbender) to infiltrate the event aided by the Basterds and German film star and double agent, Bridget von Hammersmark (Diane Kruger). Hicox and two of the German-born Basterds (Wicki (Gedeon Burkhard) and Stiglitz (Til Schweiger)) meet with von Hammersmark at a tavern. Coincidentally, a Gestapo Major (the same Gestapo Officer present earlier at Goebbels' meeting) is present, and so are a squad of German soldiers who are celebrating the birth of one of their comrades' son. One of the German soldiers strike up a conversation with Hicox, and notices Hicox's "unusual" accent, which the Gestapo Major also takes notice of. When Hicox gives himself away while ordering three glasses of Scotch by displaying the wrong three-fingered gesture (the American/British gesture with index, middle, and ring fingers, instead of the traditional mainland European gesture with thumb, index, and middle fingers), the Gestapo Major realizes the deception. The resulting standoff between Hicox, the Gestapo Major and Stiglitz erupts into a firefight, leaving everyone in the tavern dead and only von Hammersmark wounded but alive. A suspicious Raine interrogates von Hammersmark and, when she tells him Hitler himself will be attending the premiere, devises a plan for himself and two of his remaining men to attend the premiere by posing as von Hammersmark's Italian entourage. Meanwhile, Landa and his men investigate the scene of the fire-fight, he finds two clues leading directly to von Hammersmark.
At the premiere, Landa asks to see von Hammersmark privately. Confirming that she is in league with the Basterds, he fatally strangles her and orders Raine and Utivich (B. J. Novak) arrested, and taken to a separate location for interrogation with Landa. Landa makes a deal with Raine's commanding officer (Harvey Keitel) to allow Donny and Omar—still seated in the cinema—to kill the Nazi high command, in exchange for a full military pension, American citizenship, credit for the assassination and freedom from his war crimes. During the film, Zoller goes to the projection room to flirt with Shosanna again, and reacts badly after she incessantly rejects his advances. She then asks him to shut the door, and as he goes to do so, she shoots him. However, he manages to shoot her before succumbing to his wounds. The showing of Zoller's film is then interrupted by an inserted close-up of Shosanna informing the audience that they are going to be killed by a Jew. At the same time, Shosanna's employee and lover, Marcel (Jacky Ido), who has locked and bolted all the exits, ignites the nitrate film stacked behind the screen. Omar and Donowitz kill Goebbels and Hitler, then shoot into the crowd of panicking Nazis until the timers on their bombs go off and destroy the cinema.
Landa and his radio operator drive Raine and Utivich to the American lines, where Landa surrenders to Raine and hands over his weapons, allowing Utivich to handcuff him. To Landa's shock, Raine breaks part of the deal by shooting the radio operator and orders Utivich to scalp the dead man. Raine then carves a swastika into Landa's forehead, proclaiming, "This just might be my masterpiece", and the film ends.
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