This film opens with mass in a Boston Catholic church, where Irish American fraternal twin brothers Connor McManus (Sean Patrick Flanery) and Murphy McManus (Norman Reedus) pray while a sermon is read, mentioning Kitty Genovese, a real-life crime victim brutally murdered while her neighbors watched without intervening. As the priest begins his homily, the brothers approach the altar and kiss the feet of a crucifix. They depart as the priest reminds the congregation that they should fear not just evil but also the "indifference of good men". The brothers conclude that the priest finally understands, Connor stating, "I do believe the Monsignor's finally got the point..." and Murphy replying, "Aye".
Connor and Murphy work at a local meatpacking plant. While celebrating St. Patrick's Day in a neighborhood bar, three Russian "soldiers", led by Ivan Checkov, enter and order everyone to leave, as their organization has decided to evict the pub. In the ensuing bar brawl, Connor, Murphy, and the patrons publicly humiliate the mobsters, who the next day ambush the brothers in their home. As Murphy is dragged into a nearby alley to be executed, Connor escapes to the roof and drops a toilet, along with himself, off the roof and onto the mobsters, killing them and rescuing Murphy.
The Russian mob's involvement summons FBI agent Paul Smecker (Willem Dafoe) to the murder case, and he surmises that the mobsters' death was not a professional hit but self-defense. As the police begin a manhunt for the killers, Connor and Murphy arrive at the police station to clear their names. During the initial interrogation, the brothers impress Smecker with their multilingualism (including Gaelic, Russian, French, Spanish, Italian, and German) while recalling the specifics of the barfight and subsequent self-defense the next morning. Smecker believes their story and allows them to stay overnight in a holding cell to evade the press. That night, Connor and Murphy receive a vision from God telling them to destroy all that is evil so that which is good may flourish.
The brothers resolve to rid Boston of "evil men" with the help of their friend and former mob package boy David "The Funnyman" Della Rocco (David Della Rocco). The brothers trade in the weapons and valuables stolen from the mobsters' bodies for their own, and use Connor's knowledge of Russian to locate a meeting between Russian syndicate bosses at a local hotel. Crashing into the room through an overhead air duct, the brothers draw their guns and fire, killing the 8 underbosses. Forcing the leader, Yuri 'Big Man' Petrova (Victor Pedtrchenko) to his knees, the brothers recite a short prayer:
"And shepherds we shall be, for Thee, my Lord, for Thee. Power hath descended forth from Thy hand, That our feet may swiftly carry out Thy command. So we shall flow a river forth to Thee And teeming with souls shall it ever be. In Nomine Patris, et Filii, et Spirits Sancti."
The two then execute him, placing pennies over his eye sockets, in which both of his eyes were shot out. While the brothers are preparing to leave the hotel, they are surprised to find Rocco dressed as a bellboy knocking at the door with a tray of food. They decided "to fuck with him" and pull him into the hotel, concealing their identity with masks, and threaten to execute him. Eventually they reveal their identity to Rocco, who decides to join the brothers in their mission. Once again, Agent Smecker is brought to the front of the murder case. The fact that all the killings involved the Russian Mob leads Smecker to the theory that the executions were the result of the feud between the Russian and Italian mobs.
The following day, the brothers try to convince Rocco that he was sold out by Italian mob boss Papa Joe Yakavetta (Carlo Rota), because he was sent to the hotel with a six-shooter .38 revolver despite the fact there were nine mobsters in the hotel room. Rocco soon realizes this after killing two of Yakavetta's men at a deli later on, who hint to Rocco that he really was sold out. In retaliation, Rocco and the brothers hunt down and kill Vincenzo Lipazzi (Ron Jeremy), underboss of the Yakavetta crime family at a local strip club. Also killed at the Strip Club are two street criminals with no connection to organized crime (thus disproving Smecker's mob war theory). The three vigilantes proceed on a series of increasingly violent missions, cleansing the city of vicious criminals and others who have eluded justice. Papa Joe, believing that the mob killings are an act of revenge from Rocco for setting him up to be killed, contracts the infamous contract killer Il Duce (Billy Connolly), to deal with the vengeful package boy.
Rocco insist that he and the brothers murder a hitman that Rocco had briefly worked with. According to Rocco, the man was responsible for murdering an entire family and had burnt their remains in a dumpster. They head to the hitman's house and murder all of his men. After a hand-to-hand fight, Rocco beats the hitman to death with a cue ball. As they leave the hitman's house through the front door, the trio is ambushed by Il Duce, and in the resulting shootout the trio manage to chase Il Duce away. Rocco's finger is shot off and each of the brothers receive serious, but not mortal, gunshot wounds which they cauterize with a hot iron.
Hours later at the crime scene, Smecker discovers the finger and secretly takes it to conduct his own investigation. Discovering that it belongs to Rocco, whom he previously met, Smecker begins to unravel the mystery surrounding the various murders. His sympathy for the brothers conflicting with his professional desire to bring them to justice, Smecker (a flamboyant homosexual) goes on a drinking binge at a gay bar before seeking advice from a nearby Catholic church. Bemoaning the futility of the courts that fail to punish evil men and his uncertainty with the MacManus brothers' actions in a confessional, Smecker is oblivious to the fact that Rocco, who has tracked Smecker to the church, is forcefully directing the priest's responses to preserve the Boondock Saints' identities. Connor sees Rocco follow the priest into the confessional and, disgusted with the blasphemy, pulls Rocco's head through the other confessional at gun point. In whispered tones, Rocco tries to explain to Connor the circumstance while still holding his gun to the priest's head. Smecker is advised, reluctantly by the priest, that the Saint's are acting as messengers from God and that "the laws of God are greater than the laws of man." Inspired by the advice, Smecker decides to help the brothers.
The McManus brothers and Rocco infiltrate the Yakavetta headquarters to finish off the family, but are captured by Papa Joe and his henchmen who recently arrived to protect Papa Joe from such an attack. Papa Joe executes Rocco to intimidate the brothers. The brothers manage to free themselves and tend to Rocco while Smecker, disguised as a female prostitute, distracts the other mobsters. Smecker kills the remaining mobsters only to be knocked unconscious by Il Duce who mistakes Smecker for a woman. As the brothers say their family prayer over Rocco, Il Duce arrives and sneaks up behind them.
As he hears them recite the family prayer, and upon seeing that the man he was hired to kill (Rocco) is dead, he lowers his weapons and joins them. It becomes apparent that Il Duce is their long-lost father, as the brothers had previously refused to teach Rocco the prayer because it is only passed down in their family. He then joins them in their mission to rid the city of evildoers.
Three months later Papa Joe is sent to trial, and though there seems to be enough evidence to convict him, the reporters on scene anticipate his acquittal due to his Gotti-esque charisma. The trial is forcibly interrupted when the two brothers and Il Duce, aided by Agent Smecker and several police officers, infiltrate and lock down the courtroom. The three then publicly declare their mission to destroy evil and recite their prayer one last time, killing Papa Joe with several bullets (and a shotgun blast) to the back of the head. The media dubs the three "Saints", and the movie ends with various "man-on-the-street" interviews in which various Boston citizens reflect on the question "Are the Saints ultimately good, or evil?"