This is based on opinion. Please don't list it on a work's trope example list.
Nightmare Fuel / The Megas
The fact that Dr. Light is singing I Want To Be The One To Watch You Die.
Afraid of the Dark is about Shadow Man consciously losing his former personality to darkness after Dr. Wily repairs and reprograms him—and he's terrified. At around 3 and a half minutes in, the darkness sings to him.
The Haystack Principle gets nightmarish when Needle Man starts singing about how much he loathes and fears his new body, while another voice repeatedly confirms that this is who he is.
The prelude to "I'm Not the Breakman", called simply "The Red Song", can be this without much context for it. It's a wordless audio track of Mega Man running to the site he'll meet the mysterious Break Man. And then he hears something in the distance, beyond the churning machinery. A song, sounding as if it's being played on a chime. A machine explodes as the blue hero's opponent teleports in, Break Man's systems powering up as a raging fire can be heard.
Gamma. His first conclusion upon activation is to kill everything that's not a machine because that's the only way to ensure peace.
The barely suppressed rage in Gemini Man's voice at the end of his spoken narration in GeminEye, which is shortly after followed by a gunshot.
"But I looked to the stars and I swore: If we were both taking the big sleep, I'd take the rat out myself."
Out of Time, a cover of the final level music for the first Castlevania, covers the very real threat of Dracula's revival, ending on a chilling delivery of the chorus line's final verse as the music halts.
"Tonight, the lord of darkness shall. Rise."
The lines "There's something here" and "This can't be real" underscore how surreal and paranoia-inducing Castlevania can be, even for Simon Belmont.