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Several of the Wario-Man microgames in Twisted have Nightmare Fuel elements. One requires you to align Wario's spine, complete with cricking sounds. Another commands you to INCH! towards an apple as an earthworm with Wario's head. And the dancing boss stage features a Wario... THING with disembodied eyes and a face that appears to consist of Gag Nose and mustache.
One of Dr. Crygor's minigames in Touched involves you spinning a black hole so that it swallows stars and planets, including Earth.
Ashley's after-boss-microgame-break after completing her stage shows her petting her stuffed bunny and asking you to do so. Doing so causes the bunnie's HEAD to fall off.
In Smooth Moves, in the minigame where you have to pick up the controller and hold it to your ear like a telephone, usually you get a simple little message from the controller saying in a calm voice "Oh, I'm sorry, wrong number." But on rare occasions you'll hear a panicking man screaming into your ear "HELP ME PLEASE HELP ME!!"
When you fail Tiny Wario's boss stage, "Wario Dance Company", the other three dancers know when you mess up, WITHOUT EVEN HAVING TO LOOK AT YOU.
In Orbulon's Stage in Smooth Moves, one involves a juggling bear. If you fail she gives you a very creepy snarl as two large spiders appear.
In "Swat Team" from Mona's stage, if the player fails to swat the fly, it will fly into the camera, covering it completely, along with a loud buzz.
In Penny's stage, the microgame "Local News" has the player trying to keep the mic on the interviewed character's mouth. Not doing so will make them angry (the man from the 1st stage, and the polar bear from the 2nd stage get very creepy, but the samurai granny in the 3rd stage is too funny to be scary).
In the final stage of "Soiled Reputation", a dinosaur skeleton rises from the earth. Sometimes it will suddenly get its head close to the camera and take a bite.
D.I.Y. & D.I.Y. Showcase
Violent microgames made in D.I.Y can be disturbing, especially when you hear Wario laughing his ass off after clearing each one. Imagine chainsawing someone's head off only to hear Wario's "WA HA HA!" upon completion.
Game & Wario
Don't deny it, 5-Volt can really scare the crap of you in Game & Wario's Gamer stage, especially when she jumps out of the TV and into 9-Volts Room.
She will break the window to peek into the room on higher difficulties. Is she trying to make sure her son is asleep, or awake?
To make matters worse, this stage is appearing in Super Smash Bros. for Wii U and you have to hide from 5-Volt's line-of-sight (via ducking into the shadowed areas) but if you're caught, you get stunned before being launched.