According to the complaint, NFL teams operated with a $123 million salary cap in 2010 even though the season was played without a spending limit under the terms of a collective bargaining agreement that expired after the season.
"When the rules are broken in a way that hurts the game, we have an obligation to act," NFLPA Executive Director DeMaurice Smith said in a statement.
"We cannot standby when we now know that the owners conspired to collude."
The complaint cites John Mara, owner of the New York Giants and chair of the NFL Management Council Executive Committee, as publicly confirming the league directed teams to restrict players' salaries during the uncapped year.
The NFL said there was no collusion and that claims made by the players union are totally unfounded.
"The filing of these claims is prohibited by the Collective Bargaining Agreement and separately by an agreement signed by the players' attorneys last August," said NFL spokesman Greg Aiello.
"The claims have absolutely no merit and we fully expect them to be dismissed."