The statement refers to the Super League judgement from the European Court of Justice last December, which required FIFA and other governing bodies to exercise their regulatory functions in a way that is transparent, objective, non-discriminatory and proportionate.
FIFA's conduct over the calendar, they allege, "falls well short of these requirements".
PFA chief executive Maheta Molango said on Tuesday: "Legal action is the unfortunate but inevitable consequence of major stakeholders within the game - the leagues and the players - being ignored.
"What unites us is the impact that decisions about the fixture calendar, made by international governing bodies, are having. In England, we've already seen major changes to the structure of the FA Cup which have essentially been forced by the expansion of international competitions.
"Those changes affect all our members and their careers. It's the knock-on impact we've been warning about for a long time.
"Even since initial legal action began last month, led by the PFA with the French and Italian player unions, next summer's African Cup of Nations has had to be moved. That is an unavoidable result of FIFA scheduling its expanded Club World Cup for next June and July.
"AFCON will instead be played in the middle of the European season. That will directly and unexpectedly impact national leagues, clubs and - most importantly from our perspective - players. These are big, potentially career-altering decisions being made without proper consultation or negotiation.
"It's just not tenable to continue to argue that this approach to the fixture calendar is working. As always, it's the players who are expected to bend. As we have seen, eventually they will break. It has to stop."
Unions and leagues wrote to FIFA in May warning they were seeking legal advice, and last month the PFA, along with FIFPRO Europe and French players' union UNFP, launched an action in the Belgian courts to ultimately determine whether FIFA's actions had breached players' rights under EU law.
The new complaint has been joined by LaLiga, which is not a member of European Leagues. The EFL and the Scottish Professional Football League (SPFL) are among the European Leagues' membership.
The Premier League has not commented separately regarding Tuesday's complaint, but its chief executive Richard Masters did address the issue at an event hosted by FIFPRO in May.
"The problem is real. We're starting to see the impact of decisions made by regional and international bodies. The calendar is getting less harmonious with every decision that is being made," he said.
"There is no faith anything is going to change. If you feel like you are not being listened to, you get frustrated. We feel enough is enough - it is a shame we have got here."
FIFA's secretary general Mattias Grafstrom defended his organisation's position in a letter to the World Leagues Association and FIFPRO in May.
He said FIFA was "like any other competition organiser" and "fully within our rights to set the parameters of our competitions whilst respecting the regulatory framework in place".
Grafstrom added in his letter that FIFA, even with an expanded Club World Cup, was responsible for only a "fractional amount" of the total elite club games in a year, with a similarly fractional number of players involved, and offered to hold further talks with the leagues and unions over the summer.
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