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.
Also, whether it's a multiple personality or not, is Billy a genuine shy nice guy, or a passive-aggressive Nice Guy (TM) who, while less openly misogynistic than Captain Hammer, is not really any more interested in her as a person? It's a good story either way, but a very different story.
Ear Worm: The songs are — dare we say — horribly catchy.
Ensemble Darkhorse: A literal case here with the Thoroughbred of Sin himself, Bad Horse; despite only featuring in two short songs and having a brief physical cameo at the end, just about everyone loves Bad Horse.
Evil Is Sexy: The aforementioned "Slipping." Irrepressibly catchy tune? Check. Haunting melody? Check. A voice that brings to mind a master of seduction? Check to the eight-millionth power.
On the commentary, Maurissa Tancharoen says, "This is my favorite scene" when NPH grabs her chin.
Captain Hammer's groupies switch to worshiping Dr. Horrible in the end, although there's a healthy component of fear there too.
Fashion Victim Villain: Apparently, silly costumes are a requirement for the Evil League of Evil.
Faux Symbolism: Dr. Horrible's overlarge armchair in which he plots world domination. Either he feels inadequate or it's just for comedic effect.
Foe Yay: Between Captain Hammer and Dr. Horrible, who seem to take their relationship very seriously:
Dr. Horrible mentions at the very beginning of his blog that Captain Hammer is his nemesis, not some poser in a parka. Captain Hammer apparently returns the feeling as he admits in his song that everyone's villains aren't as cool as his.
"These... Are not the hammer." One act later, Dr. Horrible taunts him with "Hammer, meet nail!" If that's not a Metaphorgotten, it's a very revealing Freudian Slip.
Hype Backlash: Can a modest 45-minute humorous short really live up to the massive praise heaped upon it by its fans? Some say yes.
Internet Backdraft: Whatever you do, don't start a discussion on whether Penny's fate was a standard-issue Stuffed into the Fridge death, or a deconstruction of said concept.
Magnificent Bastard: Doctor Horrible evolves into this as the story progresses.
Misaimed Fandom: Some people, as highlighted in this essay, are arguing that a large part of the audience seems to have missed the point especially in the way it tackled the idea of Women In Refrigerators and of Billy as a Nice Guy. Billy's complete transformation into the Dr. Horrible persona is clearly and unambiguously portrayed as a tragedy; not only because of the loss of Penny, but because it costs him the very humanity that his friend associates with. Some people are still going to prefer the Well-Intentioned Extremist Dr. Horrible over the nebbish Stalker with a Crush Billy.
Moral Event Horizon: Captain Hammer dating and sleeping with Penny just because he knows it'll hurt Billy/Dr. Horrible. He might be a superhero, but this moment makes it clear he's not in it because he's a good person.
Tear Jerker: (Dr. Horrible) [singing in Act III] "And I am fine." (The look on his face and the camera's pull-away show he's anything but. He's hurting and alone.) and [singing at the end of Act III] "And I won't feel..." [the door slams shut] - (Billy) "...a thing."
"Still Alive"...
"It's okay...Captain Hammer will save us."
The Woobie: Not only does Dr. Horrible get roughed up on a regular basis but takes it in stride ("...honestly, I'll live."), he suffers the emotional equivalent of a punch to the gut several times. "Penny's Song" establishes Penny as this to some extent, although she's far too optimistic to let it stick.