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. Not to mention their robotic motions when they win...
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 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, then ride over his spine. 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.
Don't forget Dry Bones' losing animation. His skull comes apart and then fixes itself.
The new Bowser's Castle course features a gigantic and terrifying flaming rock version of Bowser, which punches a forked road in the course repeatedly. And there's a chance that it may crush you under its immense fist, depending on which part of the road you choose.
Twisted Mansion's music is very dark and intense, as it takes place in a Haunted House.