Mystery, Inc. drifts apart as the thrill of solving mysteries fades, tiresome monsters turning out to be everyday people in costumes. Daphne Blake, Mary Kay Bergman, teams up with Fred Jones, Frank Welker, to launch a successful television series that aims to hunt down a real ghost rather than a fake one, hoping to recapture the spark that brought them together in the first place.
Sometime later, Fred reaches out to Velma Dinkley, B.J. Ward, Shaggy Rogers, Billy West, and Scooby-Doo, Scott Innes, inviting the old crew to reunite for Daphne’s birthday. The plan evolves into a cross-country road trip as the gang scouts haunted locations across the United States for Daphne’s show, only to stumble into more fake monsters along the way.
Their path leads them to New Orleans, where Lena Dupree, Tara Strong, invites them to Moonscar Island, a place said to be haunted by the ghost of its pirate namesake Morgan Moonscar. Skeptical but curious, the team accepts. On the island they meet ferryman Jacques, Jim Cummings; Lena’s employer Simone Lenoir, Adrienne Barbeau, who lives in a grand mansion on a pepper plantation; and Simone’s gardener Beau, Cam Clarke.
That night, Shaggy and Scooby cross paths with Moonscar’s ghost, which somehow becomes a zombie, while the rest of the gang receives eerie ghostly warnings to leave. Despite the warnings, they decide to stay the night. The next morning, the group and Beau are chased by the zombie horde; Velma suspects Beau, while Fred and Daphne manage to trap one zombie, only to discover it’s genuinely undead when Fred tears off its head and confirms the threat.
In a dark cave, Shaggy and Scooby uncover wax voodoo dolls that resemble Fred, Velma, and Daphne, and, unknowingly, gain the power to influence their friends until a disturbance—bats in the caves—splits them apart. The others discover a hidden passage in the house that Lena claims leads to where the zombies dragged Simone away, but Velma sees the truth: there are footprints, not drag marks. The passage opens into a secret chamber used for voodoo rituals, and the group is briefly trapped after Lena’s deception is exposed.
Simone and Lena reveal themselves as immortal werecats, descended from a group of settlers who worshiped a cat god. When Moonscar and his crew attacked the island, the settlers were driven into the bayou and devoured by alligators, but Simone and Lena escaped. They cursed Moonscar and his pirates, granting themselves immortality, but at a terrible cost: the ritual requires victims to sustain their power. Jacques joined their scheme to secure his own immortality in exchange for helping them lure victims, while the zombies and ghosts on the island are the island’s former victims who awaken every harvest moon to frighten intruders away.
As the werecats close in, Shaggy and Scooby manage to disrupt the draining ceremony, giving the others a chance to break free. The werecats close ranks, only to realize too late that the harvest moon has passed; they disintegrate to dust, and the trapped zombies’ souls are finally at rest. Beau is revealed to be an undercover police officer sent to investigate disappearances on Moonscar Island, adding a note of order to the chaotic night.
Daphne, ever practical and ambitious, invites Beau to guest star on her show, hoping to showcase his skills, and by dawn the group parts ways, each heading toward a new chapter after a night of mysteries on Moonscar Island.
