Microsoft has been fined 280.5m euros ($357m; £194m) by the European Commission for failing to comply with an anti-competition ruling.
Microsoft says it has worked hard to meet the commission's demands
The software giant will appeal against the fine which follows a long-running dispute between it and EU regulators.
The move follows a landmark EU ruling in 2004, which ordered the US firm to provide rivals with information about its Windows operating system.
EU regulators also warned Microsoft it could face new fines of 3m euros a day.
Content and clarity
The daily fines will come into force from 31 July if Microsoft fails to supply "complete and accurate" technical information to rival developers, the EU said.
Microsoft's general counsel, Brad Smith, insisted the company had met the commission's demands, and said it would appeal the decision.
"This is not about compliance, this is about clarity," Mr Smith explained during a conference call.
Mr Smith said Microsoft would argue that the commission's original demand was too vague, and that the company had since done everything it could to comply with the ruling and provide all the information that was required.
Microsoft expected to deliver the final bundle of information for use by rival software firms on 18 July, he said, well ahead of the EC's deadline of July 24.
"Hopefully we will bring this chapter to a close in the next couple of weeks," Mr Smith said, adding that while Microsoft was not happy about the fine, the company had seen signs of progress in resolving the dispute.
EU Competition Commissioner Neelie Kroes said she had "no alternative but to levy penalty payments" against Microsoft, adding that "no company is above the law".
"I regret that, more than two years after the decision... Microsoft has still not put an end to its illegal conduct," Ms Kroes said.
Brussels had warned Microsoft in December that it would face fines of 2m euros a day if the firm failed to meet the commission's demands.
Under the 2004 ruling, Microsoft was told to provide rival firms with more information about its software, in order to enable them to write programs that could run more smoothly on Microsoft's widely-used Windows operating system.
The judgment also called for Microsoft to debundle its Windows Media Player from its Windows operating system, and slapped the software firm with a record fine of 497m euros.