Thou Shalt Not Kill: Personally against murder, which he sees as beneath him—or at least, would like to. It seems to be implied that the real reason he won't kill is because he knows it's wrong, but he doesn't wanna admit that, so makes up an excuse about it not being "elegant or creative" enough.
Neat Freak: Why he constantly wears gloves. Pay attention to him during the "My Eyes" song and it becomes clear he's hiding it from Penny. Possibly just an extension of his superiority issues over everyone else.
Not Listening to Me, Are You?: Her speech to Billy in act I trails off into "...so they can buy rocket packs and go to the moon... and become florists" when she sees that he's not paying any attention.
Small Role, Big Impact: Though he only has a seconds-long cameo at the very end of the video, Bad Horse's high standards for joining the Evil League of Evil is what drives Dr. Horrible to build his freeze ray and set off the plot.
The Unintelligible: Subverted. Speaks only in horse sounds for most of the ELE comic, with Jefferson claiming that Bad Horse was appointing him "vice president." Subverted at the very end when Bad Horse speaks English.
Professor Normal: A cyborg with a super strong robotic arm and jaw attachment. Otherwise, he's a normal person with a wife, kids, and a mortgage, hence his name.
Tie-Die: A hippie-themed supervillainess able to make hippies violent and angry.
Dead Bowie: A David Bowie-inspired supervillain with the power of persuasion. Claims to be the real David Bowie who was murdered in 1980 and that every other appearance by Bowie afterward was an impostor.
Fury Leika: Her name is a pun off of "Hell hath no fury like a Woman Scorned." She was left at the altar and then pooped on by a radioactive dove. She gained poisonous flowers, incendiary rice, a ball and chain around her ankle, and four henchwomen each carrying something old, something new, something borrowed, and something blue.
Fake Thomas Jefferson: Claims to be the real Thomas Jefferson, though that is unlikely unless Jefferson had Super Strength. Also carries a quill pen tipped with tranquilizer. Doesn't like to have lots of stuff in his pockets.
Snake Bite: A supervillainess with a cobra-themed costume. Carries a staff topped by a mini disco ball. Did not appear in the "Evil League of Evil" comic with the other ELE leaders.
Hourglass: A villainess who is able to see the future. Tells Moist about a kid who will become President. An unseen character.
Bait and Switch: A pair of villains who Moist goes on a double date with. Somehow able to fool him into thinking he was dating Bait instead of Switch. Bait is apparently the more attractive of the two.
Conflict Diamond: A villain friend of Dr. Horrible and Moist. Went on the double date with Moist, Bait, and Switch.
Pink Pummeler: A homophobic villain with Super Strength, Gaydar, and a superhuman knowledge of Broadway show tunes. Repeatedly rejected by the ELE due to his tendency to date rather than beat up his victims.
Purple Pimp: A supervillain who appears at the party for Dr. Horrible's induction into the ELE.
Johnny Snow: An amateur hero not affiliated with the Council of Champions. He wears a parka and carries an Ice Beam. He's mentioned in Act I and appears in the ELE comics, where he foils a plot by the ELE to poison the city's water supply by freezing it. The villains are impressed and mistake him for a supervillain.
James Flames: A potbellied superhero with pyrokinetic powers. His Catch Phrase is "M'name's James and I have the power of flames." Wears a singed T-shirt and pink overalls. He thaws the water supply after Johnny Snow freezes it.
Wingspan: A member of the Council of Champions, along with Captain Hammer. Works in a command center in the sky. He, along with the rest of the Council, foolishly leaves the city for a retreat, allowing the ELE to wreak havoc. Appears briefly in the ELE comic.
Elementia: A blue-skinned superheroine and a member of the Council of Champions. Works in a command center with a waterfall and apparently has some kind of Elemental Powers. Has no problem sitting naked in a sauna with men.
???: An unnamed member of the Council of Champions with a third eye in the middle of his forehead. He appears in the sauna with the rest in the ELE comic.