This is based on opinion. Please don't list it on a work's trope example list.
Nightmare Fuel / Mario Kart 8
Metal Mario and Pink Gold Peach having blank sclera for eyes.
When entering slow-motion, various sounds such as doing tricks off of a ramp or earning/losing coins can come off as creepy and sinister.
Most of Koopa Troopa's voice samples sound... off. They can get borderline psychotic when he places 1st or executes a mini-turbo or boost.
Unagi the Eel from Super Mario 64 returns in the track Dolphin Shoals. The good news? He looks much cartoonier, and less inherently creepy. The bad news? He's twice as big, and his massive body forms part of the track, so you need to drive straight at his face. It almost fills the screen.
Dry Bowser's victory animation is rather creepy. He moves slightly to the side and shakes violently for a second. Repeatedly.