Microsoft was fined €280.5m on Wednesday for failing to comply with an anti-competition ruling.
The 2004 ruling imposed a record fine of €497m and ordered the software giant to provide rival firms with more information about Windows.
Microsoft now stands accused of failure to obey the Commission, which said that the firm could face daily fines of €3m if it fails to supply "complete and accurate" technical information to rival developers.
Redmond denies that the fine will have any effect on the launch of its next-generation Windows operating system, scheduled for release to business customers in November before a general release by January.
Microsoft chairman Bill Gates said this week that there was an "80 per cent chance" that Vista would be ready to ship as scheduled by January 2007.
"I don't think that the fine has any impact on the teams and the efforts that are going forward to that launch," Darren Huston, president and chief executive of Microsoft Japan, told a news conference. "We are still committed to the existing plan of record."
In a statement about the fine, Microsoft general counsel Brad Smith said: " We have great respect for the Commission and this process, but we do not believe that any fine, let alone a fine of this magnitude, is appropriate."
Smith confirmed that Microsoft will be taking the case to the European courts.
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