FIFA president Gianni Infantino wants to expand the World Cup to 48 teams starting with the 2026 tournament, with Asian countries already backing the controversial plan.
Manchester City boss Pep Guardiola warned last week that the global saturation of football was going to "kill" players and the European Club Association (ECA) added its criticism, saying it had sent a letter to Infantino opposing the moves for a bigger World Cup.
"We have to focus on the sport again," said ECA chairman Karl-Heinz Rummenigge, who is also chairman of Bayern Munich.
"Politics and commerce should not be the exclusive priority in football.
"In the interest of the fans and the players, we urge FIFA not to increase the number of World Cup participants."
The ECA, an independent body representing football clubs at European level, said that the number of games every year "has already reached an unacceptable level".
Founding members of the ECA include Manchester United, Chelsea, Real Madrid, Barcelona, Juventus, AC Milan and Bayern.