Earlier this month, Fifa president Gianni Infantino proposed a 48-team tournament consisting of 16 groups of three countries each.
The European Club Association (ECA) say the number of games played in a year is already at an "unacceptable level".
"We urge Fifa not to increase the number of World Cup participants," said ECA chairman Karl-Heinz Rummenigge.
Fifa's council will discuss Infantino's proposal at a meeting on 9 January but the 46-year-old made expansion part of his election manifesto because he wants to allow more countries the chance to compete at football's flagship event.
The number of teams competing at a World Cup last increased in 1998 when the tournament expanded from 24 to 32 countries but any change to the current structure would not be likely to take effect before the 2026 World Cup.
Rummenigge added: "We have to focus on the sport again. Politics and commerce should not be the exclusive priority in football."
The ECA represents more than 200 clubs, including Real Madrid, Barcelona, Juventus, Bayern Munich, Manchester United and Chelsea.
The organisation representing Europe's leading clubs has rejected calls for the World Cup to be expanded.