Mr Van Rompuy, who could chair the key meetings about an independent Scotland's EU membership, made his comments on YouTube.
Scottish Labour seized on the remarks as further proof of what it regards as the SNP's folly of trying to break up the 300-year-old United Kingdom.
Mr Van Rompuy was taking part in a Q&A with an internet user called Keith, from Edinburgh. The video emerged following the row over whether a newly-independent nation would automatically have to apply for membership.
Mr Van Rompuy was asked if he agreed with Scottish nationalism and the "evil work it portrays to end the United Kingdom?"
He replied: "Nobody has anything to gain from separatism in the world of today, which, whether one likes it or not, is globalised. We have so many important challenges to take and we will only succeed if we can pool forces, join action, take common directions.
"The global financial crisis is hitting us hard; climate change is threatening the planet. How can separatism help? The word of the future is union."
Shadow Scottish Secretary Margaret Curran said: "Scots will rightly be asking the same question that Mr Van Rompuy did – is separation in this global age really the way forward?
"We agree with the European Council president in his belief that the best way to tackle the problems we face is by binding together, not breaking apart. I would suggest that Alex Salmond stops his assertions and listens to those who know more about this than him.
"When you also look at the reports that Cyprus will want Scotland to join the queue to join the EU, the SNP's claims that Scotland will simply walk straight into the EU are shown to be more and more ridiculous by the day."
The Nationalists claimed the EC leader's words were "completely out of date" as they were posted in June 2011. The SNP insisted that because of growing eurosceptism at Westminster, a yes vote in the 2014 referendum was the only real way to secure Scotland's future in the EU.
Asked if Mr Van Rompuy was still opposed to separatism, his spokesman told The Herald: "I would expect that his views would not have changed; he may be a bit more cautious as it has become a more salient issue."
But a source close to the EC president made clear he would not be campaigning against Scottish independence and added: "Scotland will need to reapply for EU membership and he could chair the meetings where that is discussed. I can imagine some nations would make that a difficult meeting – Spain, Cyprus and Belgium."
Alyn Smith, the SNP MEP, branded the highlighting of Mr Van Rompuy's remarks "completely out of date" and pointed to the Edinburgh agreement, which, he said, for the first time had produced an agreed, legal process by which Scotland could become an independent country.
"It is the Tories who are threatening Scotland's place in Europe," declared Mr Smith. "We want to remain part of the EU but increasingly clearly London doesn't.
"Why should Scotland leave such an important issue to Tories obsessed with isolating the UK from the rest of Europe?"
He pointed to claims by Graham
Avery, an honorary director general of the EC, that a newly independent Scotland would be fast-tracked.
Jose Manuel Barroso, President of the European Commission, has claimed Scotland as "a new state" would have to apply for EU membership, as has Spain's Foreign Minister Jose Manuel Garcia-Margallo.
Meanwhile, Stewart Hosie, the SNP's Treasury spokesman, has suggested the Bank of England would be just as much Scotland's central bank as England's after independence.
But Scottish Labour leader Johann Lamont said the idea was "dishonest or delusional."