The movie starts off with Batman and Robin plummeting into action, trying to stop Mr. Freeze from stealing a very large diamond. A battle ensues, with Batman and Robin pitted against Mr. Freeze and his henchmen at a museum. The Bat-Duo fails to stop Mr. Freeze when Robin gets ahead of himself and ends up getting frozen by Mr. Freeze, allowing him to escape while Batman tries to thaw Robin.
Meanwhile, Dr. Pamela Isley, a botanist, discovers that her boss, Dr. Woodrue, is using her venoms to create a criminal called Bane. He kills her for discovering this secret; however, she survives the poisons that are intoxicating her and kills Woodrue after she reawakens.
Back at the Batcave, Batman and Robin are doing research on Mr. Freeze. Alfred has begun to show signs of fatigue, apparently due to his old age. The next day, Alfred’s niece, Barbara Wilson, drops by on a surprise visit. The day after that, Bruce unveils a new powerful telescope for which he has donated the funds. This is where Pamela Isley forces her way up to ask Bruce for funding, which he rejects. Later, Bruce decides to conduct a diamond auction in an attempt to lure Mr. Freeze into a trap. However, Poison Ivy comes in and mesmerizes everyone with her spores. Mr. Freeze comes in to steal the diamond, and with Poison Ivy there already, it distracts Batman and Robin from capturing Mr. Freeze. However, the duo go after him anyway. Batman disables Robin’s bike from jumping a bridge. Batman jumps the bridge himself and is able to land on Mr. Freeze and arrest him. Back at the Batcave, Robin gets into an argument about trust with Batman.
While Dick is doing some research in the Batcave, he catches Barbara sneaking out with a motorcycle and decides to follow her this time. He follows her to an underground motorcycle race, and participates himself. But he ends up saving Barbara from falling off the bridge when the biker gang trips her. Back at the mansion, Barbara reveals to Dick that she wants to take Alfred away from his life of servitude. Bruce then reveals to Dick that Alfred is dying.
Meanwhile, at Arkham Asylum, Poison Ivy breaks in to free Mr. Freeze in order for them to team up. Dick then alerts Bruce that Mr. Freeze has escaped, and the Bat-Duo rushes over to Mr. Freeze’s former hideout. Batman and Robin go deeper into Mr. Freeze’s hideout, where they encounter Bane and Poison Ivy. The Bat-Duo ends up fighting each other instead, giving Ivy and Bane enough time to escape. When Ivy returns to her own hideout, she tells Mr. Freeze that Batman killed his wife, when in fact it was Ivy who did it. Back at the mansion, the doctor reveals that Alfred has stage 1 of MacGreggor’s syndrome, which is the same stage to which Freeze already has the cure. Later, after Alfred gives Barbara a CD containing secrets and telling her to give it to Alfred’s brother, Barbara tries to hack into it to see what it is. She is eventually successful and discovers the secrets about Batman and Robin.
Later that night, Bruce is finally able to convince Dick to trust him before he goes to see Poison Ivy. Meanwhile, Mr. Freeze plans to take over the telescope that Bruce Wayne had donated in order to freeze the entire city. Barbara eventually finds her way down to the Batcave, where Alfred has already tailored a Bat-costume for her. Robin goes to meet Poison Ivy, who tries to poison him by kissing him. However, Robin has trusted Batman, and he had a rubber cover over his lips. Batman drops in to help him but all the vines tangle them up. Eventually, Batgirl comes and rescues the Bat-Duo.
Afterwards, the three of them rush over to try and stop Mr. Freeze. All three of them eventually fight their way to Mr. Freeze, whose plan they foil. They are able to thaw the city by using the satellites to reflect the sun towards the city. Batman also succeeds in obtaining a cure for Alfred from Mr. Freeze, and afterwards they use it on Alfred. The next morning Alfred awakens and seems to be healthy again. The film ends with Poison Ivy hallucinating inside her cell in Arkham Asylum.