Often, mistranslations of the older games have reintroduced the words in their archaic meanings. For an example, one of the persons listed in the credits of the NES [Famicom] game Hi no Tori: Wagaou no Bouken is called "Nice Gay Masami".
In Banjo-Kazooie, during the board game challenge, Gruntilda calls you a "furry geek". Neither word is by itself unambiguous, but put them together...
In one scene in L.A. Noire, the main characters comment on an item related to the movie "Gay Cowboys". This may be a reference to Brokeback Mountain, though in the game's time period the title is perfectly innocuous.
In the description of the legalize gay marriage edict for Tropico, your advisor uses the old definition of gay, thinking that the new law would allow happy people to get married.
Done quite deliberately in the ZX Spectrum text adventure Bored of the Rings (which may or may not have had some connection with the book of that name), which at one point had Fordo (the protagonist) meet up with a few "manly" (that was exactly how it was put, quotes and all) pop stars of the day. Stick around there too long, and you got "Fordo felt a queer sensation" followed by his accidentally dying of AIDS a few turns later.
Deliberately lampshaded in Two Worlds II: Modern English is the language of all characters except for one, who goes for extremely exaggerated Middle English, leaving the game's hero wondering what exactly he'd just heard. At one point, the odd character starts talking about his experiences with leading a "gay life" - i.e., having a happy existence. Shown in about 70th second of this video.
Very early in BioShock Infinite, you will come across a barbershop quartet giving a performance of "God Only Knows". There are signs nearby that proclaim the group as, among other things, "Columbia's Gayest Quartet".
One target in Assassin's Creed IV: Black Flag tells his minions to leave no bush unmolested. He basically means "leave no stone unturned" but one suspects Abstergo will censor that part.
In the first Baldur's Gate game, one of Imoen's selection quotes is "You're a queer fellow".
When confronted by a giant pink cat boss in Shadow Hearts: Covenant, protagonist Yuri exclaims "That's one big pussy!"
In the "Harvest Scramble" DLC for Fire Emblem Awakening, a character (who is also a boss in a side-mission in the game) directly references and says that they're "Having a gay old time".
Moraff's Dungeons of the Unforgiven (a 3D but turn-based graphic adventure, which didn't do very well because it had the misfortune to come out at about the same time as Wolfenstein 3D) had "Lesdidian" warriors. Yes, they were female.
The description for the homosexual trait in Crusader Kings II says "this character is a bit queer..."