the release of virtual boy

The cutscene after the water dungeon in Twilight princess... That was creepy as all hell.

I remember when I got scared by that huge eel in Jolly Roger Bay the first time I played Mario 64...

The first 10 months of 2016 when we had a new NX rumour every single day while Nintendo kept us in the dark and refused to just come out and reveal the fucking thing. At a time when it seemed they'd completely lost the plot, a lot of the downright terrible rumours seemed frighteningly plausible.

When your Switch freezes and memories flash before your eyes because you might lose that 200-hour BotW save file.

 and 

A lot of stuff already mentioned. The eel in Super Mario 64, redeads and wallmasters in Ocarina of Time, a lot of stuff in Eternal Darkness, phanto in Super Mario Bros 2...

A few more to add

This fella in DK64 (was in DKC2 if I recall correctly).


This guy used to freak me out as a kid too.

Third party, but the first person sections in Jurassic Park on the SNES scared me as a kid as well.

Why do they SCREAM.

When they announced Nintendo Switch Online I was most frightened.

Beat me to it by several days.