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)
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. 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.