The appearance of the first zombie: a skeleton is half poking out of the wall of a pit into which Shaggy and Scooby have fallen, then an ethereal green light enters it, causing it to wrench itself out of the wall and regrow rotting flesh. The fact that it's an angry pirate (with a rusty cutlass, no less) doesn't help matters.
Simone's, Lena's, and Jacques' transformations
Daphne: You won't get away with this.
Simone: I've been getting away with it for 200 years! (cue transformation)
Their deaths too. We see them howl in agony as they sizzle and fry. We get to see the fur be fried off of the skeletons, which then crumble to dust.
The final deaths of the poor zombies as well, though this is more bittersweet considering their suffering is finally over. They literally decay, and the flesh is literally torn from their bodies.
During this whole scene, the most powerful, nightmarish music you can imagine is playing, appropriate for the breathtaking end of such a powerful nightmare. Seriously, how did the gang ever fall asleep afterwards?
The scene where Shaggy and Scooby are nearly drained of their life force by the cat-women.
Fred's, Daphne's, Velma's, and Beau's faces melting after their wax voodoo dolls get too close to a torch. You can literally see the life leaving them as they melt. Thank goodness Scooby and Shaggy knocked the dolls away in time.
The part where Fred tries to prove the zombie isn't real:
Fred: *trying to pull the "mask" off* It's the gardener!
Fred: *keeps pulling* It's the fisherman.
Shaggy: Like, no.
Fred: *keeps pulling" It's the Ferry Man.
Fred: Well, maybe it's- *pulls the head clean off* ... real!
To make matters worse the head starts to move a lot. Scaring them more.
"Who opened a window?!"
The flash back revealing Simone and Lena's back story, specifically when the pirates force their family and friends into the water to be eaten alivebyalligators. Doesn't justify their horrific actions, but one can't help but sympathize at least a little for their tragic past and their desire (though it becomes truly monstrous) to exact revenge on the pirates.
The moans of the zombies sound pretty ethereal, and very inhuman - especially when we first meet the undead Morgan Moonscar. Becomes even more terrifying when later it's revealed that this inhuman moaning and groaning is literally the undead's desperate attempts to save the gang from suffering their fate.
The sheer number of zombies shows just how many poor people had their life drained by Simone, Lena, and Jacques.
The entire "It's Terror Time Again" sequence. This was a chase scene unlike anything before in the franchise. The usually cheerful gang who've never been in such peril are running for their lives from a horde of real undead monsters. Never before have the gang looked so terrified. And the epic song by Skycycle is an aggressive (and AWESOME) assault on the senses to really hammer it home; after all these years, it's terror time again!
The revelation that there really is a malevolent, or at the very least vengeful and capricous cat-God in existence that was partly behind Simone's reign of terror.
The poster itself is pretty scary. It's big in scope, with the heroes looking on at something, terrified and on the run in a spooky bayou. Meanwhile, some truly frightening looking zombies are overlooking the poster from the sky, and we also finally have a scary gothic castle in the distance in front of the full moon. note While one may think that the castle will be The Very Definitely Final Dungeon, it's only featured in the prologue Is this a poster for a Big Damn Movie or what?