Produced by the same creative team as The New Batman Adventures and Batman Beyond, the movie appears in the same timeline, appearing between the two, as evidenced by the presence of the same Robin. A new hero has arrived in Gotham, but as the title suggests, her identity is a mystery; even to Batman. Batman must figure out who Batwoman is and to stop familiar enemies Penguin and Rupert Thorne from selling illegal weapons to the fictional nation of Kasnia. At the beginning of the story, the two employ Carlton Duquesne, a gangster, to provide protection. Bane also features later on in the story as additional muscle.
Batwoman's main focus is on illegal activity by the Penguin, Thorne, and Carlton Duquesne. Despite taking the symbol of the Bat as a sign of justice, Batwoman sullies the Bat prefix by taking out criminals with ruthless and dangerous techniques. She seems uninterested in sparing the lives of her adversaries.
Batman, with Robin, sets out to stop the Batwoman from making mistakes as she tries to take out the villains, and as he encounters numerous twists, setbacks, and apparent false leads in determining her true identity. The newest gadget on display is a wind glider used by Batwoman that utilizes some of the most advanced technology ever seen in Gotham City. Bruce Wayne, Batman's alter ego also becomes involved with a new lady in his life: Kathy Duquesne, the crime boss' daughter.
In addition to Kathy Duquesne, Bruce is introduced to two other women who, as his investigation continues into who the Batwoman is, seem to fall well into suspicion: Roxanne "Rocky" Ballantine, a new employee of Wayne Tech whose technology development is used by the Batwoman against the Penquin; and by Detective Bullocks new partner Sonya Alcana, whose knowledge of the weapons being smuggled by the Penguin and Carlton Dunquesne is much greater than the detective should know.
With Carlton Dunquesne unable to stop Batwoman's raids on the various facilities used to hold the various weapons, Penguin calls Bane for additional support to ensure that there are no more losses as a result of the Batwoman. Not long after Banes arrival in Gotham, it is revealed that there is not one but three Batwomen, all of whom were the women suspected by Batman. They had taken turns to remove suspicion on any one of the three, while using Roxannes technological genius and contempt for the Penguin (who had framed her long-time boyfriend), Kathy's money and access to several key aspects of her fathers organization, and Sonyas physical and police skills to ensure that Penguins operation was thwarted.
- Kevin Conroy as Bruce Wayne / Batman
- Efrem Zimbalist, Jr. as Alfred Pennyworth
- Tara Strong as Barbara Gordon
- Bob Hastings as Commissioner Jim Gordon
- Robert Costanzo as Detective Harvey Bullock
- David Ogden Stiers as Oswald Cobblepot / The Penguin
- Eli Marienthal as Tim Drake / Robin
- John Vernon as Rupert Thorne
- Hector Elizondo as Bane
- Kevin Michael Richardson as Carlton Duquesne
- Kelly Ripa as Dr. Roxanne "Rocky" Ballantine
- Kimberly Brooks as Kathleen "Kathy" Duquesne
- Elisa Gabrielli as Detective Sonia Alcana
- Kyra Sedgwick as Voice of Batwoman
- Cherie as Iceberg Lounge Singer
- Barbara Gordon
- Alfred Pennyworth
- Commissioner James Gordon
- The Penguin
- Batwoman / Rocky
- Batwoman / Kathy
- Batwoman / Sonia
- Rupert Thorne
- Detective Harvey Bullock
- Carlton Duquesne
- Jay, Raven, and Lark