The second special costume mode gives Ashley a full plate armor, whose helmet noisily closes as you aim at her. Have fun watching the enemies as they try to grab the girl and fall hopelessly to the ground due to her heavy armor. Or Leon himself whenever he has to catch her.
Using the PRL-412 on the Final Boss without charging it has amusing results.
Sometimes when Leon kills an enemy while escorting Ashley, she'll do a cute little fist pump in victory.
Jump off a ladder (without catching Ashley) and aim up at her, and she'll call you a pervert and cover up her skirt.
Probably unintentional on the designers' part, but if Leon gets knocked on his back by an enemy near Ashley and happens to be positioned right, she'll similarly react as if he's trying to look up her skirt. It's hysterical to hear Ashley chastising Leon for being a pervert while he's getting his brains bashed in!
Made even better when Leon actually dies under her, and she calls him a pervert even as the game fades into the "You Are Dead" screen.
In the original Gamecube version, there is a bug that allows Ashley to Suplex certain enemies if you quickly press A after stunning them by opening a door in their face. It's pretty hilarious AND can make that part of the game easier.
When you first rescue Luis in Chapter 1:
Luis: ...Okay. I have one, very important question... do you got a smoke? Leon:(shrugs) ...I got gum...
A moment of unintended hilarity can happen with Luis if you're wearing Leon's first alternate costume:
In the island military base, there's an oven containing a Ganado. Most first-time players won't know what to do (and take double-damage if the fan-nicknamed "Oven Man" grabs them), but those expecting should step back immediately as soon as Oven Man bursts through. He dies of his own burns shortly.
Every time a Ganado blows himself up with his own dynamite like he's friggin' Wily E. Coyote.
The fact that one of the Ganados steals Leon's jacket for no reason.
Of course there's a good reason. That was a nice jacket.
The Laser-Guided Karma punishment for shooting Luis multiple times. On the last bullet, the game stops, and plays a cutscene where Luis yells "Adios, Leon!" and nails him with two bullets from his own gun.
Playing as Leon you get a few humorous comments if you look at items. Playing as Ada? It's dialed up to Aya Brea levels.
"Is this what passes for police work these days?" On finding the officer in the village.