Superior Court of Sporting Justice officials said on Friday that the decision meant 2012 champions Fluminense would go down to the second division instead next season.
Last month the court docked four points from Portuguesa and Flamengo for fielding suspended players.
That decision saved Fluminense from relegation after they had originally finished two points behind Portuguesa.
The court also reinstated Flamengo with the docked points on Friday but their top-flight status was not at risk.
The Brazilian Football Confederation is now expected to appeal against the latest rulings. The new season starts in May.
"This (issue) does a disservice to Brazilian football," the court said in a statement.
"It generates uncertainty and instability for the organisation of competitions, causes serious financial losses to clubs and fans, without mentioning the effect it has on Brazil's credibility on the eve of the World Cup."
The World Cup in Brazil begins in June.
The court added the original punishments breached the Supporters Statute that rules the league must publish its decisions online for them to become applicable.
The clubs had said they were not properly informed of the player suspensions.
(Editing by Tony Jimenez) nL3N0KK4WZ
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