Players union International Federation of Professional Footballers (FIFPRO) filed a legal claim against world football's governing body FIFA, it said on Thursday.
In May, FIFA said they would not consider rescheduling their 32-team Club World Cup after global players' union FIFPRO and the World Leagues Association (WLA) threatened legal action if they did not review their plans.
FIFPRO said England's Professional Footballers' Association (PFA) and France's players union (UNFP), both of which are members, submitted a legal claim with the Brussels court of commerce on Thursday.
"FIFPRO Europe member unions have today submitted a legal claim against FIFA, challenging the legality of FIFA's decisions to unilaterally set the International Match Calendar and, in particular, the decision to create and schedule the FIFA Club World Cup 2025," FIFPRO said in a statement.
"Player unions believe that these decisions violate the rights of players and their unions under the EU Charter of Fundamental Rights while also potentially violating EU competition law."
FIFA did not immediately respond to an emailed request for comment from Reuters.
In May, FIFPRO and WLA had expressed their concern over the expanded competition in a letter addressed to FIFA president Gianni Infantino and secretary general Mattias Grafstrom.
Their letter said the global football calendar is "beyond saturation" and that national leagues are unable to properly organise their competitions, while players are being pushed beyond their limits, with significant injury risks.
In response, FIFA rejected their claims that it had made unilateral decisions to benefit its competitions in the international calendar.