The film opens at Kasumi's Temple, where she is arguing with Hayabusa about her brother, Hayate's, supposed death. She understands that if she leaves to find out what happened to her brother, she will become a shinobi, and her army will kill her. Before leaving the grounds of the temple, she is confronted by Ayane (Natassia Malthe). Kasumi (Devon Aoki) acknowledges that Ayane is in love with Hayate, and promises to find out what happened to him. She jumps over the temple wall, and catches a flying shuriken as she falls - on which the words "You are invited DOA" are inscribed.
Out at sea, Tina (Jaime Pressly) is arguing with her father about professional wrestling on her yacht. A gang of pirates underestimate her, and try to take her boat. Tina comprehensively defeats them, and a DOA shuriken pierces the side of her boat. In a Hong Kong hotel, Christie (Holly Valance) is being investigated by a number of policemen after a theft of diamonds. Only in a towel, she manages to disarm and defeat all of them, and then leaves the building, driving off on a motorcycle, receiving her own shuriken. Back at Kasumi's palace, Ayane is instructed to follow Kasumi, and kill her for betraying the clan.
Kasumi, Christie and Tina decide they must work together to reach the top of the compound by sundown. The three girls are amongst a number of invited fighters on a plane going to DOA island. Maximillian Marsh, who left Christie at the hotel to face the police alone is also on the plane, as is Hayabusa, as well as Tina's father (Bass) and Zack, who mocks Tina, saying wrestling isn't really fighting. On an in-flight screen, Helena (Sarah Carter) welcomes them all as the best fighters in their chosen fields, and tells them they now must parachute the rest of the way, and whoever fails to make it to the main compound by sundown will be disqualified. Tina, Christie and Kasumi all land well off target under a tall pagoda structure, and after realizing they may not make it in time, grudgingly combine their efforts to climb up it to the main citadel. As they reach the top, Kasumi momentarily sees Ayane's face, realizing that she has been followed to DOA. The fighters go on to meet Donovan (Eric Roberts), the organizer of this year's DOA, who explains about the $10 million prize money, and some of the history about the tournament.
Kasumi fights Leon. Each fighter is analyzed in a lab to show their strength, and are all injected with nanobots which record their abilities. Weatherby, who operates all the technical equipment, oversees all this. After all fighters are issued with ID bracelets, which beep and show the name of your next opponent in the tournament, the confrontations soon begin, with Tina, Christie, Gen Fu, Hayabusa, Helena, Bayman, Zack all progressing to round two. Meanwhile, Kasumi recalls her brother, and how he once saved her from ruthless kidnappers. He fought with precision and skill, far beyond any warrior Kasumi had ever known. After saving her, she remembers Hayate's face when a DOA shuriken came to him. Before her first round fight, Kasumi visits Donovan, who explains that Hayate was defeated by Leon the previous year, falling from the top of a cliff with his body never to be found. After some more reflection, Kasumi finds herself also matched against Leon; however, she soon defeats him, realizing that if she can win so easily, Leon could not possibly have beaten Hayate.
Some time later, Hayabusa and Kasumi have both progressed to the semi-final stage of the tournament, and all combatants take a time-out to let off steam. A volleyball tournament takes place down on the beach, with Kasumi and Tina against Helena and Christie. Meanwhile, Hayabusa explores Donovan's compound whilst all eyes are on the game, but is soon captured by the building's booby traps. At the beach, the game abruptly ends when the ball is struck down by a throwing star, and Kasumi realizes that Ayane is calling her out. They fight each other in the bamboo treetops, and Kasumi pleads for Ayane's help to find Hayate, pointing out that when he deserted the clan, she never pursued him out of love. Ayane coldly replies that he was different, and that Kasumi should come to terms with his death. Their confrontation ends with the arrival of the other girls, prompting Ayane to flee.
Christie and Helena engage in an epic quarter-final fight. With the recreation time over, the tournament resumes with Tina being matched against Zack. The two fight at the forbidden square, and Tina manages to defeat him. Zack apologies for mocking her, finally acknowledging that she is a true fighter, much to Tina's appreciation. The result leaves a showdown between Helena and Christie. Before the fight, Max and Christie discuss how a source had said "Helena is the key" to finding the Douglas vault somewhere on the island, inside which is over $100 million. In a dramatic beach fight in the rain, Christie notices a tattoo on the back of Helena's neck. Despite being caught off guard by the image, Christie still manages to overpower Helena, leaving her lying unconscious on the wet sand, and progressing to the semi-finals of the tournament in her own right. Returning to Max, she recalls and deciphers the cryptic image, showing the location of the vault to be in the Buddha head statue. With a new round beginning, Tina, Christie and Kasumi wonder where Hayabusa has gone, and go looking for him at Donovan's compound. However, they themselves fall into a trap, and are also captured.
It turns out that Donovan is planning a terrible scheme to harvest the nanobots from the four best DOA fighters, creating a technology where the host will be able to predict and adapt to any fighting style and defeat any combatant. The technology had been Weatherby's, who had no idea Donovan would use it against fighters' wills, as well as for profit. He reveals to Helena that her father had objected to the idea, and on that very night he was killed. After being comforted by Weatherby about her failure against Christie, Helena gains new resolve to stop Donovan. Fighting through all his security forces, the two make it to the compound and prevent Donovan from broadcasting the technology to buyers around the world. Meanwhile, Max finds the Douglas vault and breaks the code, but is interrupted by Bayman, who under Donovan's command knocks out Max and takes the cash.
The girls join forces to defeat Donovan. Despite being able to stop the transmission, Donovan manages to download the DOA programme onto specialist sunglasses, making him unstoppable. He reveals that he captured Hayate a year ago, and much to everyone's surprise, frees him. He challenges Hayate to one fight, in which the technology allows him to effectively predict every move Hayate makes. The distraction, however, buys Weatherby time to start unlocking the harvest pods to free the four semi-finalists, and Helena also manages to hold off Donovan for a small time. Donovan, however, gets the upper hand, taking a hard copy of the technology and setting the entire compound to self-destruct. With all the fighters freed, the girls join forces to take on Donovan as he attempts to escape in a four-on-one fight. Meanwhile, Hayabusa fends off Bayman with the help of Weatherby and Max, and Hayate is aided by Ayane. After a tough fight, Christie manages to knock off Donovan's glasses, removing his power of supremacy. Kasumi and Hayate then finish him off, immobilizing him with ninja skill. In the final moments, and the compound about to explode, all the combatants jump off the cliffside to the water, whilst Weatherby, Max and Hayabusa take an escape hatch.
Managing to gain a boat (humorously from the same pirates that attacked Tina earlier in the movie), the group all together escape DOA island. Back at Kasumi's palace, the five female fighters join forces against the hundreds of warrior guards
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