The European Commission's action on Tuesday came after a year-long investigation following complaints by Dutch Olympic speed skaters Mark Tuitert and Niels Kerstholt.
The skaters said the sport's governing body had threatened to bar athletes from ISU events if they compete in big-money Ice Derby events run by a South Korean company.
The Commission said such rules restrict athletes' commercial freedom and prevent new entrants from organising alternative international speed skating events as they are unable to attract top athletes.
"We have concerns that the penalties the ISU imposes on skaters through its eligibility rules are not aimed at preserving high standards in sport but rather serve to maintain the ISU's control over speed skating," European Competition Commissioner Margrethe Vestager said in a statement.
The Commission said a system of penalties revised by the ISU in June, which range from five-year to lifetime banks, remains disproportionately punitive.
The ISU said the EU charges were unfounded.
"A neoliberal and deregulated approach to sport could destroy the Olympic values underpinning sport," the governing body said on its website.
Ben Van Rompuy, legal adviser for the two skaters, said the EU action was a loud and clear signal to other sports bodies with similar disproportionate restrictions on athletes.
"Sports federations cannot abuse that regulatory role to block or restrict, without any valid justification, other operators from organising sports events and athletes from pursuing much-needed economic opportunities," Van Rompuy said.
Tuitert said on Twitter: "Sports governing bodies should act truly responsible and not abuse their power."
The EU executive will consider the ISU's reply to its charge sheet, known as a statement of objections, before it issues a ruling.
(Editing by Ed Osmond)
