Batman and Robin attempt to stop Mr. Freeze from stealing a cache of diamonds. The supercriminal freezes Robin and flees, notifiying Batman that he has 11 minutes to thaw his partner. They learn that Freeze was once scientist Victor Fries, who became dependent on a diamond-powered subzero suit following an accident in a cryogenics lab he was occupying to find a cure for his wife, Nora, who was suffering from a terminal illness called MacGregor's Syndrome.
Meanwhile, botanist Dr. Pamela Isley is experimenting with the strength serum "Venom" to create mutant plants capable of fighting back against mankind's destruction of flora. She is furious to learn that her senior colleague Dr. Jason Woodrue used her Venom samples to transform a diminutive prisoner into the "super soldier" Bane. When she refuses to become the mad scientist's partner, Woodrue kills her with animal-plant toxins and chemicals. She transforms into the beautiful Poison Ivy and kills Woodrue with a venomous kiss before vowing to use her new powers to establish botanical supremacy over the world.
Alfred Pennyworth's niece Barbara Wilson makes a surprise visit from England and is invited to stay at Wayne Manor until she returns to university. Alfred is revealed to be suffering from MacGregor's Syndrome.
Ivy arrives in Gotham City with Bane as her henchman. She interrupts a Wayne Enterprises press conference at the Gotham Observatory where a giant new telescope is being unveiled, exhorting Bruce Wayne to implement a proposal that would safeguard the natural environment at the expense of millions of human lives, which Bruce summarily declines. She later appears at the Gotham Botanical Gardens fundraiser, seducing everyone present with her pheromone dust, including the Dynamic Duo, who are there to protect a diamond on auction from Mr. Freeze. When Freeze crashes the event as expected, Ivy is instantly captivated by his "ruthless charm". Despite being captured by Batman and detained at the Arkham Asylum, Mr. Freeze is released by Ivy.
Ivy then proceeds to turn off Nora Fries' life support and makes Mr. Freeze believe Batman did it, thereby persuading him that they should kill Batman as well as the society that created him. They plan to turn the observatory's new telescope into a giant freezing gun to freeze the whole world's population to death and replace them with Ivy's mutant plants.
Meanwhile, Robin has become infatuated with Ivy and rebels against Batman, just as the villainess planned. He eventually goes to meet her at her garden hide-out. Ivy's venomous kiss fails to kill Robin because Batman had prevailed on him to coat his lips with rubber. She tries to drown Robin in her lily pond and entangles Batman in her crushing vines, although they are able to free themselves when Batgirl arrives and traps Ivy in her own floral throne. Having discovered the Batcave and decided to create her own crime-fighting persona with Alfred's help, Batgirl reveals herself as Barbara.
When the Trio arrive at the Observatory to stop Freeze, the criminal has already frozen Gotham. Bane attacks Robin and Batgirl, but they incapacitate him and restore him to his original human state. The two younger heroes save Gotham by repositioning the observatory's satellites, using their mirrors to reflect sunlight from outer space to thaw the city.
Batman shows Freeze video footage of Ivy's confession about pulling the plug on Nora, but reveals that he himself restored Nora. He vows that Freeze will be allowed to continue his research at Arkham Asylum to heal Nora. Batman asks Freeze for the cure he created for the first stage of MacGregor's Syndrome for Alfred and Freeze atones for his misdeeds by giving him two vials of the medicine he had developed.
At Arkham, Ivy is joined in her cell by Freeze, who promises to make her life a living hell for attempting to kill his wife. Back at Wayne Manor, Alfred is cured and Bruce invites Barbara to live with them and continue helping him and Dick fight crime as Batgirl.