"It is totally unacceptable that a representative of the U.S. administration criticized an independent court of law outside its jurisdiction," Kroes told reporters.
"The European Commission does not pass judgment on rulings by U.S. courts, and we expect the same degree of respect."
Kroes's statement raised the stakes in a split over the Microsoft case between the Bush administration and the European Commission.
The Commission has a history of cooperation with the U.S. Federal Trade Commission and the Justice Department since patching up differences over an EU decision in 2001 to block General Electric's takeover of Honeywell.
On Monday, the CFI in Luxembourg upheld a landmark 2004 Commission decision that the U.S. giant abused the near-monopoly power of its Windows operating system to damage competitors, along with a 497 million euro ($695 million) fine.
Kroes has defended the order by her predecessor, Mario Monti, that Microsoftsell a version of Windows without its Media Player audiovisual software, and share information to enable rival makers of servers to operate smoothly with Windows.
Barnett, the Justice Department's top antitrust official, disparaged the EU's policy.
"We are ... concerned that the standard applied to unilateral conduct by the CFI, rather than helping consumers, may have the unfortunate consequence of harming consumers by chilling innovation and discouraging competition," he said in a written statement on Monday.
Barnett said the United States protects competition, not competitors.
Some of Microsoft's main U.S. competitors complained to the European Commission. Other U.S. rivals sparked a similar antitrust case against Microsoft in the United States by complaining to the Justice Department in the 1990s.
A U.S. Court of Appeals found in 2001 that Microsoft violated the Sherman Act antitrust law. Since then, some states have criticized the Justice Department for backing off.
Kroes said Microsoft may appeal against the EU decision.
"If the parties to a case are unhappy with the Court of First Instance ruling, they can appeal to the Court of Justice, and that is well known by those parties," she said.
Microsoft has not said if it will do so.