Coach Z of Homestar Runner was shown married to Bubs and has worn a skirt and hair curlers in two different toons. The only reason why he is ambiguous is because his sexuality is never mentioned. He's likely Ambiguously Bi due to his obsessive attraction to Marzipan.
Barry 'The Blender' Henderson and Thomas 'The Tanker' Smythe of I Am Fighter both fit this trope to some extent; Thomas frequently takes it to Squick levels.
Red vs. Blue: Donut. In Season 1, he had been given pink armor ("It's not pink, it's lightish red!") and was your typical new recruit. In Season 2 (where this trope comes into play) he started using lotions, talking about his feelings, screaming like a woman, analysing dreams, and generally being as effeminate as possible while retaining some air of heterosexuality. It's gone by Season 3.
Tucker: I don't think Santa's outfit is a biker's costume and a codpiece, Donut. Donut: It was the best Christmas ever!
The song "Donut: The Musical" from the Season 9 soundtrack plays it straight. The song is full double-entendres and similar sounding words (that the other Red members keep asking to repeat). And he still thinks he can get some "elbow grease" and "headlight fluid".
"Let me blow you... away"
In Yu-Gi-Oh! The Abridged Series, Pegasus is an even stronger example of this trope than the original. He even goes so far as to violently defend this image, as once the side characters find out that Pegasus had a wife, he knocks them unconscious with the words "I can't have people thinking I'm straight".
Bakura, to the point where his own personalities can't agree on the issue.
Bakura: I'm not gay, I'm just British! Yami Bakura: I'm not British, I'm just gay.
Marik is either in a Transparent Closet or out and dating Yami Bakura, although it's less ambiguous and more Marik failing to even realise he's gay.
Marik: Oh, what, just because a guy likes to dress effeminately and hang around with another extremely attractive man and read yaoi and flaunt his gorgeous abs and stroke a phallic symbol suggestively in every other scene, that automatically makes him gay?! Bakura: ...Kinda.
Dragon Ball Abridged plays Zarbon's ambiguous sexuality for as many jokes as it can. Soon before his death however it is revealed, to the surprise of everyone, that he is completely straight.
Jean-Armand (codename: Nephandus) of the Whateley Universe. Obsessed with his looks and his hair and his clothes. Chaka even refers to his standard outfits as a 'Merchant-Ivory wardrobe'. A mama's boy, although his mother is a notorious European supervillain called the Troll Bride. Carries a walking stick (although it's a weapon too). Even his friends in the Bad Seeds make jokes about his ambiguity.
Omega Zell from Noob is a reporter for a feminine channel in real life, cares for his avatar's appearence over anything else, is frequently forced into an Involuntary Dance and acts as if he has a crush on the game's (male) top player. His Yaoi Fangirl guildmate speculated he could be gay from day 1, but he has always denied it. Season 4 has him reveal that he's deliberately avoiding relationships with women to be able to focus on his dream of becoming the next top player of the game (that and he sort of hates them). However, neither the series creator nor the fans seem able to stop making jokes about him having a crush on any male character whose actor ends up in close proximity to him on "behind the scenes" photos.