But it slightly reduced the fine from 899m euros ($1.12bn; Â£719m) to 860m euros.
The fine was imposed four years ago and was a record at the time.
It was imposed because Microsoft had not provided certain information about its products to competitors as it had been ordered to do in 2004.
EU anti-trust regulators originally fined Microsoft 497m euros in 2004 for abusing its dominant market position to block competitors.
Microsoft was ordered to make the code for its server software available to competitors so their products could work alongside it.
In 2008, Microsoft was given an additional fine for failing to comply with the 2004 order.
The latest decision, "rejects all the arguments put forward by Microsoft in support of annulment", a statement from the EU General Court court in Luxembourg said.
At a hearing in May 2011, Microsoft's lawyers had argued that the fine was excessive and undeserved.
Europe's second highest court has upheld a competition ruling against Microsoft saying it "essentially upholds the Commission's decision".