Technically, yes. The real question is if the game supports multiple gamepads.
If the game didn't support them, pressing a B button on both controllers would execute the associated action for both players at the same time
Can't recall the name, but there was a game I played on PC, featuring some kind of local co-op/versus mode, which had this issue.
Most of them, however, usually work flawlessly. As GmanBOSS said, just make sure that they're both connected before you launch the game, as most games usually won't recognize new input devices on the fly.