During the hands, these characters provide humorous chatter between each other and towards the player. GLaDOS (voiced by Ellen McLain) from the Portal series takes a supporting role as the dealer. (voiced by Patrick Warburton), Ash Williams from The Evil Dead franchise (voiced by Danny Webber), and Claptrap from the Borderlands series (voiced by David Eddings). The four additional characters in addition to the player are Sam from the Sam & Max franchise (voiced by David Nowlin), who is assisted by Max (voiced by David Boat), Brock Samson from The Venture Bros. Like the original game, there is a large amount of dialogue due to the banter that the characters share. In addition to the Texas hold 'em style of play featured in the original game, Poker Night 2 introduced the Omaha hold 'em style as a playable mode as well.

Each player starts with the same amount of virtual money (which was raised to $20,000 per player and $100,000 total from $10,000 per player and $50,000 total in the original game), and competes in standard poker rules to try to eliminate all the other players by exhausting their money. Like the original game, Poker Night 2 is a computer-based poker simulation between an unseen silent participant (the player) and four characters that is played in The Inventory that was featured in the previous game. See also: Gameplay of Poker Night at the Inventory

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