As a child, Bruce Wayne falls down a dry well, where he is attacked by a swarm of bats; he subsequently develops chiroptophobia. While watching an opera with his parents, Bruce is frightened by performers masquerading as bats, and asks to leave. Outside, the family is targeted by a mugger, Joe Chill, who murders Bruce's parents, leaving him to be raised by the family butler, Alfred Pennyworth.
Batman et Robin sont invités à une démonstration très secrète animée par le professeur Hugo Strange et son assistant, le Dr Harleen Quinzel, qui pourrait éventuellement changer à jamais le futur de Gotham. En cours de route, Batman s'arrête au pénitencier d'État de Gotham pour rendre visite à Catwoman (dont la tentative de suicide du premier film a échoué) pour lui remettre un livre de poésie d'Elizabeth Barrett Browning. C'est quelque chose qui la tiendra occupée pendant les 36 prochains mois de sa vie jusqu'à ce qu'elle soit définitivement libérée de prison. Au laboratoire, ils rencontrent le procureur et allié de Bruce Wayne dans la guerre contre le crime Harvey Dent, célèbre pour avoir arrêté un gang de contrefacteurs de pièces tout en conservant un quartier à deux faces en souvenir de l'affaire.
The Joker and the Penguin break out of Arkham Asylum and race each other to find a stash of stolen money hidden within a crypt in Gotham Cemetery after an inmate, who had hidden it, told them about it. The Joker is quickly intercepted by the Batman and is electrocuted when he falls into a river by his super-charged joy buzzers while the Penguin takes the opportunity to enter the cemetery himself. While searching through a crypt, the Penguin accidentally cuts his hand while using his umbrella-sword to open a coffin he hopes contains the money, finding a body instead. Blood from his hand drips on the corpse's heart, which brings it to life; it is none other than the vampire lord Count Dracula, his body having been moved from Transylvania to Gotham City after his "death." After attacking and turning a watchman at the cemetery into a vampire, Dracula hypnotizes the Penguin into becoming his non-vampiric servant so that he may lead him through Gotham to feed and guard his coffin during the day while he regains his strength.
During a conference of crime bosses held in a Gotham City skyscraper, gangster Chuckie Sol is killed by a mysterious cloaked figure, shortly after Batman bursts in on the meeting. Due to the killer's resemblance to Batman, the Dark Knight is blamed for Sol's death. Councilman Arthur Reeves tells the media that Batman is a public menace (despite Commissioner Gordon's protests), then later attends a party at the mansion of billionaire Bruce Wayne, Batman's secret identity. Reeves teases Bruce about his bad luck with women and for having allowed an old girlfriend, Andrea Beaumont, to get away.
Batman, Batgirl et Robin forment une alliance avec les Tortues Ninja pour affronter Shredder qui s'est apparemment joint à Ra's al Ghul et sa Ligue des Assassins, afin de pouvoir prendre le contrôle de la ville.
At a manor in Sarajevo, Ra's al Ghul ruefully realizes his mistake in allying himself with the Joker, while his assistant informs him that the Joker has captured Jason Todd, the second Robin. In a warehouse, the Joker brutally beats Jason with a crowbar while Batman races there. Joker then leaves the warehouse and traps a half-dead Jason inside. Although Jason attempts to escape, a planted explosive destroys the building before Batman can rescue him.
Batman and Robin fail to stop Mr. Freeze from stealing a cache of diamonds. They learn that Freeze was once a scientist named Victor Fries, who became dependent on a diamond-powered subzero suit following an accident in a cryogenics lab while working to save his wife, Nora, from a terminal illness called MacGregor's Syndrome.
The Dynamic Duo face off against the Wizard, a hooded villain with an electrical device which controls cars and a desire to set challenges for the Dynamic Duo, whose identity remains a mystery throughout until the end.
In Gotham City, Batman stops a hostage situation in a bank caused by Two-Face, the alter ego of the disfigured former district attorney, Harvey Dent. However, Two-Face escapes. Edward Nygma, a researcher at Wayne Enterprises, develops a device to beam television directly to a person's brain; Bruce Wayne—with whom Nygma is obsessed—rejects the invention, noting that it "raises too many questions", and Nygma angrily resigns from his position after killing his supervisor Fred Stickley, and forging his suicide note and footage. Everybody except Bruce is convinced it was a genuine suicide. During a news report, it shows how Harvey Dent became Two-Face: While he was prosecuting Sal Maroni, Maroni threw acid in Harvey's face; Batman tried to stop him but failed. After meeting Batman-obsessed psychiatrist Dr. Chase Meridian, Bruce invites her to a charity circus event. There, Two-Face and his henchmen storm the event in an attempt to discover Batman's secret identity, and in the process murder The Flying Graysons, a family of acrobats who attempt to stop him. The youngest member, Dick, survives and throws Two-Face's bomb into the river to detonate safely underwater but the rest of the family dies.
Alors qu'il combat Gorilla Grodd, Batman est pris dans sa machine temporelle et est envoyé au Japon féodal. Il y est chassé par des samouraïs au service du Joker. En s'échappant, il rencontre Catwoman, elle aussi prise dans le temps depuis deux ans, qui lui apprend que les criminels de Gotham sont tous là et sont devenus des Seigneurs féodaux après avoir abusé le daimyō. Batman et Catwoman doivent donc intervenir pour empêcher les vilains de ré-écrire l'histoire.
When Gotham is plagued by crime committed by an animal-themed villain squad composed of The Penguin, Silverback, Cheetah, Killer Croc and Man-Bat, Batman and other heroes band together to stop them.
Mister Freeze et Le Pingouin sont de retour à Gotham City. Après avoir réfrigéré la ville, le scientifique transforme Bane et Killer Croc en monstres géants dévastant tout sur leur passage. Afin de les combattre, Bruce Wayne élabore un robot géant de Batman et le combat commence. Dans sa quête pour ramener la paix, le super héros sera suivi dans son affrontement par Robin, Green Arrow, Nightwing et Flash.
It’s Halloween night in Gotham City and Scarecrow, Clayface, Silver Banshee and Solomon Grundy have hit the streets to stir up trouble! Batman is on the trail of the city’s spookiest villains while, further complicating matters, the clown prince of crime himself, The Joker, is ruling over this mysterious crew of misfit criminals. It’s up to the Dark Knight to stop this gruesome gang before they unleash “digital laughter,” a computer virus that’s part of a diabolical plan to jeopardize all of Gotham City’s vital technology. Batman, Green Arrow, Cyborg, Nightwing and Red Robin must combine forces to battle these baddies and save the city.