These are what we call the 'YMMV items.' Things that some people find in this work. We call them 'your mileage might vary' because not everyone sees these things in the same way. This starts discussions in the trope lists, a thing we don't want. Please use the discussion page if you'd like to discuss any of these items.
Anticlimax Boss: Invinciblity or otherwise, Iosa's second phase goes by quite quickly. And the first phase is just easy; she insists on patrolling back and forth below platforms conveniently located for you to jump out of reach of her guns.
Best Boss Ever: The fight with General Tor. Not only is it the most difficult and intense one in the game, it is fought at the sound of Awesome Music.
Breather Level: Sector 6. Sector 5 is an extremely long slog through a couple of very large buildings, with crossfire everywhere, your Mission Control gone and very urgent music Seven Four. Sector 6 takes things slow and steady, with small groups of weak enemies separated by stretches where you can just saunter along and enjoy the atmosphere while Iji looks for Dan, with the same peaceful music as in Sector 1, Kinda Green, playing.
Draco in Leather Pants: The Tasen in general, really. While there are a few good ones, they apparently contrast so much with the Komato that many, many fans make them out as almost saintly. While they aren't as vicious as the Komato, there's still the problem that they tried to wipe out life on Earth, and that's not touching on all their war crimes in the backstory - what they did to Iosa's planet stands out.
There's also a surprising amount of people who view Krotera as a Well-Intentioned Extremist doing whatever he needed to do to protect his people. You'd think that the game established well enough that he's a jingoistic tyrant with a 0% Approval Rating who chose to Alpha Strike Earth instead of making peace with the humans because he could.
Family-Unfriendly Aesop: Some of the Komato state the logical, if abhorrent, conclusion that since their species is doomed anyways, the Tasen's desperate attempts to avoid their fate only lead to more suffering for all involved.
Krotera makes the exact same point with regard to humans.
Game Breaker: The Null Driver is a literal example. Firing it will lead to random changes in crucial data, up to and including changing the size and shape of the level geometry. Fortunately, the changes aren't permanent. Also a straight example as well. Proper use of it allows you to fully level up all attributes by Sector 2.
Which is why it's only unlocked once everything else is, and has to be reacquired every time you start the game.
It only lasts for thirty seconds, but Explosive Shotgun buff + Buster Gun (automatic shotgun) = Fully-Automatic, Unblockable, Instant-Hit Explosive Goodness.
Goddamned Bats: Komato Skysmashers, flying turret guns that dodge around so you can't easily hit them and pack some serious firepower. When they do go down they explode, which hurts you. To quote the game creator, "No-one likes the Skysmashers."
Love to Hate: Asha is just such a colossal asshole, but he also provides some of the best fights in the game, and finally taking him down is very satisfying (or alternatively denying him his rematch and watching him lose his mind.)
Memetic Badass: reallyjoel's Dad. After a Youtube user named "reallyjoel" posted a comment on Daniel Remar's own speedrun video saying that his dad could do better, there's been a running joke that reallyjoel's Dad is the best gamer in the world, and that there's a top-secret "reallyjoel's Dad" difficulty mode. Daniel said that reallyjoel's Dad played said difficulty and beat it so hard that it was wiped from existence. However, It's still in the game, and it's as impossible as they say.
"I approach you friendly, and you are firing blue lettuce at me?"
"You promised me a pony! With missiles!"
"No missile pony for you."
Moral Event Horizon: Asha holding Dan hostage and possibly even killing him depending on the player's actions is this both out and in-universe. Even on a pacifist run, Iji has no qualms about killing Asha after this point.
Ugly Cute: Annihilators. Hell, there's an Annihilator plushie in the game.
Unintentionally Unsympathetic: Despite the game's message that "the enemy" are people too, many players found it a good deal easier to kill Komato than Tasen. It's not surprising that portraying a species whose hat is being genocidal Sociopathic Soldiers as sympathetic would be difficult, to say the least. Even Daniel Remar has remarked that he may have gone too far with how he portrayed them.
Viewer Gender Confusion: Ansaksie is mistaken for a guy fairly often. Ironically, she was specifically made female to differentiate her from Asha. Also, many players thought the diary writer to be a guy from reading her diaries, since it's all just text logs with no voice for comparison and the number of times she refers to her girlfriend outweighs the number of references to her own gender.
The Woobie: Iji, and to a lesser extent, the Diary Writer.
Tropes in the webcomic:
Anticlimax Boss: Played for Laughs. Vateilika tries to fight Iji at the ends of Sectors 3 and 6, but either way she isn't any more powerful than a regular Soldier. The first time, Iji just kicks her out the window, and the second time, she pushes her out the door.
Big Lipped Alligator Moment: At one point, during a discussion on how portal guns violate the law of conservation of energy, we cut to Dan watching Ron fall through an infinite portal loop while Kyubey freaks out in the foreground. No one acknowledges he's there, it's not brought up in the narration, and he's never mentioned again. Captain Lhurgoyf just included it as a one-off non sequitur joke.
Later, a command asks for something silly and unrelated to the plot to happen just because it's silly, which Captain Lhurgoyf renders as a monkey in a party hat, clown makeup, a tuxedo jacket, and a diaper juggling chainsaws on a unicycle while blowing a streamer in front of St. Basil's Cathedral. It even uses the Big Lipped Alligator Moment icon from The Nostalgia Critic.