Depending on the town's inhabitants, you can also have villagers commenting that other villagers of the same gender should hook up.
In City Folk, you can harvest mushrooms during the fall. Some of them resemble psilocybin containing mushrooms. The furniture you get from these also resembles various psilocybin containing mushrooms. Your character can actually eat the mushrooms that do not contain furniture.
The character Pascal. His mannerisms (and speech), seem vaguely like what you'd expect from someone using some kind of drug...
Although sometimes villagers will tell you that he was perfectly normal before an accident from playing sports occurred.
In the GCN version of Animal Crossing, Tom Nook's greeting upon entering his store includes the line "Feel free to browse, but try not to carouse!" To carouse is to drink lots of alcohol and party heartily.
In Wild World, one of the messages-in-a-bottle that you can receive (generated by the game, instead of one from another player) consists of a word puzzle with the fourth (and final) word missing. The outgoing line asks if you can guess what the final word is. The three preceeding words? Fish, Dish, Disk, Dick.
or "Dusk" or "Desk"
When you capture a Stag Beetle the protagonist makes a pun about stag parties, another term for bachelor parties.
In the German version, one of Mr. Resetti's rants conclude with him going to watch "Der Bewegte Maulwurf" (The Moved Mole), a play on "Der bewegte Mann" (The Moved Man), a famous German movie - based on a comic - about a straight man being thrust into homosexual subculture.
In some of the games you can obtain a statue of a naked mermaid.
As well as the Manekkin Pis (statue of a peeing boy) and the Venus de Milo (statue of the Roman goddess of beauty, which happens to be topless). The stark-naked Michaelangelo's David joins them in New Leaf.
Wild World makes reference to alcohol in the fly's bug menu description.
In New Leaf, you can crossdress. That doesn't necessarily fall into this trope, but what Mabel says when a male tries on a dress at the store DOES.
Mabel: Well, I guess it's okay to be adventurous sometimes.
Reading the descriptions of the fish and bugs in New Leaf goes into surprising detail about them, including mentioning that some species can change sex or are completely sexless. Not genderless, sexless. While it's completely accurate, you wouldn't expect an E rated game to use sexless over the more generally accepted but incorrect term genderless.
Kappn's song about his butt having a crack down the middle.
Again in New Leaf, there's a Double Entendre storm when a cherry salmon is caught: it mentions "cherry salmon pie".
Big Sister villagers may mention turf wars in other towns before they move there. They then mention that it's because slinging turf around sounds like fun.
One of Kapp'ns songs, he sings about getting married and singing love songs to his wife, then he sings. "I gots ta be careful what I sings about now. As a Dad there's some stuff I can't allow."
There is a magazine with the picture of a female mole in a yellow bikini on the cover lying around Resetti's Headquarters. She has a little heart next to her making it even more obvious it's an adult magazine.
In Wild World, one conversation between a Normal villager and a Lazy villager results in the Normal villager accusing the Lazy villager of being sexist. How much child-friendly media can you think of where the word "sexist" is used?