Ulysses Everett McGill (George Clooney), a suave, fast-taking convict, escapes from incarceration in Mississippi during the Great Depression. He is chained to two other prisoners, slow-witted Delmar (Tim Blake Neslon) and hot-tempered Pete (John Turturro), so the three must escape together. Everett convinces them that he has hidden $1.2 million after robbing an armored car, and promises to split it with them. They hitch a ride with an elderly blind man on a railway handcar, and he foretells that they will indeed find a treasure, though it may not be the one they seek.
They travel on foot to visit Pete's cousin, Washington Hogwallop, who removes their shackles and allows them to sleep in his barn. However, the trio is awakened by the authorities, led by the partly-blinded World War I veteran Sheriff Cooley (Daniel Von Bargen) after Hogwallop turns them in for the reward. The barn is set ablaze, but Everett, Pete, and Delmar escape with the help of Hogwallop's rambunctious young son (who drives them out of the fiery barn in a car).
They continue their journey, and encounter a religious congregation in the midst of a mass baptism. Pete and Delmar are drawn in and are baptized as well, but Everett resists. They later pick up a hitchhiking young black guitarist, Tommy Johnson (Chris Thomas King), who claims he sold his soul to the devil in exchange for musical talent. They hear that the nearby WEZY radio station pays people to sing into a can, so they pay a visit to the blind disc jockey (Stephen Root), and sing a version of "Man of Constant Sorrow" with Tommy accompanying them. Calling themselves 'The Soggy Bottom Boys', they are paid cash and leave satisfied. However, unbeknownst to them, their record becomes wildly popular around the state, with no one knowing the identity of the band.
That night, the shadowy Sheriff Cooley and police track them down and find their car near their campsite. Everett, Pete, and Delmar part ways with Tommy as they escape. The next day, they meet famed robber George 'Babyface' Nelson (Michael Badalucco), on the run from police, and accompany him in robbing a bank. He gives them a share of the stolen loot and departs.
The trio encounters three sirens: beautiful women washing clothes in the river, and are seduced by them. Delmar and Everett discover the next morning that Pete has disappeared, and Delmar believes the women had turned him into a toad (which was found in Pete's abandoned clothes). Carrying "Pete" in a shoebox, Delmar and Everett go to a restaurant where they meet Big Dan Teague (John Goodman), a one-eyed Bible salesman. Thinking that their box contains money, Big Dan lures them to a field for an advanced tutorial on salesmanship. He violently beats the two men, kills the toad after finding no cash, and steals their car.
Everett and Delmar arrive at Everett's hometown, where he attempts to speak to his estranged wife, Penny (Holly Hunter), mother of his seven daughters. He finds that Penny is engaged to Vernon T. Waldrip, the campaign manager for Homer Stokes (Wayne Duvall), who is running for governor against the grouchy elderly incumbent, Pappy O'Daniel (Charles Durning). Penny refuses to take Everett back, and was so ashamed of his arrest that she told their daughters he was hit by a train and killed.
Rejected, Everett and Delmar attend a movie, where a prison chain gang is in the audience. Pete, it turns out, was turned into the police by the Sirens, and is once again in chains. In the theater, Pete advises his friends to abandon their quest, as it is a "bushwhack."
That night, Everett and Delmar stealthily break him out of jail. Pete tearfully confesses that, after threatened with death by the authorities, he revealed their plans to find the armored car loot for Sheriff Cooley, whom they finally learn is the one who has been hunting them across the state. However, Everett reveals that he fabricated the story to entice Pete and Delmar to escape with him. Everett had truthfully been arrested for practicing law without a license, and was determined to escape when he heard his wife planned to remarry. If caught, the trio could face an additional 50 years in jail. An enraged Pete tackles Everett.
The three stumble upon a Ku Klux Klan rally in a nearby field. Shocked, they see that Tommy Johnson has been captured and that the Klan is preparing to hang him. The trio disguise themselves as color guard members and attempt to rescue Tommy, but are confronted by Big Dan Teague, a member in attendance. The Grand Wizard, it so happens, is candidate for governor Homer Stokes. After a scuffle, Everett, Pete, and Delmar topple a huge fiery crucifix onto Big Dan, presumably killing him, and escape with Tommy.
The four men arrive at a campaign dinner, disguised by long false beards. Pretending to be the hired band, they slip onstage and entertain (Delmar sings an impressive version of "In the Jailhouse Now") while Everett attempts to speak to Penny again. When the men launch into "Man of Constant Sorrow", they watch in awe as the entire audience rises to its feet and cheers, recognizing them as the elusive Soggy Bottom Boys. However, Homer Stokes arrives and tries to reveal them as the men who disrupted the lynch mob in performance of its duties. The townspeople are outraged at Homer's confessed racism, and literally ride him out on a rail. Everett, Pete, Delmar, and Tommy resume playing, and a delighted (and victorious) Pappy O'Daniel joins them onstage and grants them an official pardon. After the event, Penny takes Everett back, but demands that he return to their old cabin and retrieve her wedding ring. The four men depart quickly, as the cabin is in the valley that is due to be flooded the following day. They briefly see Babyface Nelson again, re-captured by police, but in extremely high spirits, happy at the thought of being executed in the electric chair.
The men arrive at the cabin the next morning, but to their horror find that Sheriff Cooley has caught up with them and his men have already dug their graves. As the authorities loop nooses over a tree branch, Everett drops to his knees and prays that he might see his daughters again. At that moment, the valley is flooded. The cabin is destroyed, and Everett, Pete, Delmar, and Tommy surface on the newly-made lake. They retrieve the sought-after ring from a floating rolltop desk, and return to town.
Shortly afterwards, Everett is happily reunited with Penny and his children. The family is taking a walk through the town when Penny remarks that the ring Everett brought her is the wrong one. She firmly asserts to a frustrated Everett that he must find the original ring (now at the bottom of a lake). Their daughters sing the hymn Angel Band as they cross paths with the elderly handcar operator who had predicted Everett's fate.