Microsoft rivals have filed a complaint at the European Commission, saying the firm competes unfairly and is dragging its heels over a 2004 court ruling.
Microsoft's dominant position has brought a number of complaints
They want an end to practices they say reinforce Microsoft's monopolies, and for it to comply with the court ruling forcing it to share software codes.
The group, including Oracle, IBM, Sun Microsystems, Nokia and RealNetworks, now wants EU regulators to take action.
Microsoft said the group had used litigation in the face of "innovation".
The EU's 2004 ruling said the firm had abused its dominant market position, and leaves Microsoft facing a fine of 2m euros ($2.4m; £1.64m) a day if the commission believes the software giant has not complied with the court ruling.
The ruling said Microsoft had to provide rivals with computer codes that would let them develop products to work with its Windows operating systems.
The firms who have lodged the complaint are part of a group called the European Committee for Interoperable Systems (Ecis), which was formed in 1989.
Ecis says limits placed on Microsoft after the 2004 case, and now under appeal by Microsoft, need to be fully enforced.
"Ecis deeply regrets that strong antitrust law enforcement appears to be the only way to stop the sustained anti-competitive behaviour of Microsoft," Simon Awde, chairman of the group, said in a statement.
As well as forcing it to share software Brussels also hit Microsoft with a record 497m euro fine for abusing its dominant position.
In December the European Commission said that the company was dragging its feet and not living up to its requirements.
But earlier this month Microsoft said that was not the case, and it now says that it will stand up to the fresh complaints.
"We have come to expect that as we introduce new products that benefit consumers, particularly with the kind of breakthrough technologies in Office 12 and Windows Vista, a few competitors will complain," it said in a statement.
Microsoft said IBM and some other competitors were attempting to use the regulatory process to their business advantage, and that it would respond quickly to any eventual request from the commission relating to the complaint.