The EU also said on Wednesday that it would fine Microsoft a further three million euros a day from July 31 unless it obeyed its ruling.
The ruling in 2004 ordered Microsoft to provide information that enables software developers to make products that work with its Windows operating system.
Neelie Kroes, the EU's competition commissioner, said: "Microsoft has still not put an end to its illegal conduct. I have no alternative but to levy penalty payments for this continued non-compliance."
Microsoft said it would appeal against the decision.
Brad Smith, Microsoft's general counsel, said: "We do not believe any fine, let alone a fine of this magnitude, is appropriate given the lack of clarity in the commission's original decision and our good-faith efforts over the past two years."
The EU rejected Microsoft's claims that its demands were vague and shifting.
"I don't buy that line," Kroes said, describing the EU's demands as "crystal clear".
Microsoft said it fully understood what it needed to do only after talks with the EU this spring .
The EU had already fined Microsoft 497 million euros in 2004.
The US government also ruled against the company on a similar issue in 2000, but is still awaiting technical documents from Microsoft that it ordered in 2002.