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Crowning Music of Awesome: Take your pick, there are many good tunes in this game. The soundtrack is by Jake "virt" Hoffman.
Demonic Spiders: Rare, but there are a few enemies that can really pose a problem, due to your being limited to moving and shooting in only eight directions while they have complete freedom of movement.
Goddamned Bats: Most weaker enemies until you acquire the beam they are weak to, which is thankfully early on. However, the tentacles that emerge from the red goo to shoot poison gas at you continue to be annoying throughout the game. The little floating balls of fire are also annoying, as they are often positioned to screw up your jumps.
And the evil jellybean monsters always come in enormous groups, don't self-destruct like the other basic enemies and instead keep biting you relentlessly, often hurt you even when they're dying, they're chomping is the Most Annoying Sound of the game (and you'll hear it VERY often) and they're ugly. And ANY enemy in the game can become this if you're in a tunnel, due to your movement being restricted even more.
Nightmare Fuel: Like any setting involving an invasive virus that can assimilate other lifeforms, it's there. Some of the Apocalyptic Logs provide some, but the creepiest moment comes when Jenosa confronts The Source, which has since absorbed Professor Derad, the only survivor on base whom Jenosa had any communication with. The Source speaks to Jenosa through Derad.
A lot of the bosses can be this. The final boss in its first form is almost as hellish as its final form. Due to a mini-boss that comes right before it you'll likely have lost quite some health and be contaminated to a small degree. The final boss's first form takes a loooooooooooong time to kill, so if the constant spamming of Wave Motion Guns doesn't kill you, the contamination will. You need to be able to switch between weapons lightning fast to beat this guy, all while dodging the constant rain of death falling on you when the boss's weakpoint is open. Plasma Bombs are your friends.
That One Level: Some of the upgrade-granting puzzles that require extensive use of the Adrenaline ability, as well as one switch in the Mines which requires that you freeze the more powerful and faster-thawing lizard-like creatures.
The Forest area too, mostly because it's by far the longest level in the game with the toughest enemies you'll come across.
YMMV • 
Radar • 
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Awesome) • 
Fridge • 
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Nightmare Fuel • 
Shout Out • 
Plot • 
Tear Jerker • 
Headscratchers • 
Trivia • WMG • 
Recap • 
Ho Yay • 
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Memes • 
Haiku • Laconic • 
Source • Setting