Herman Van Rompuy said a newly independent state, breaking away from an existing EU member, would be classed as a "third country" and would have to apply using "the known accession procedures".
He was responding to the announcement of an independence referendum in Catalonia.
Labour and Conservative MSPs said his comments "blew away" SNP proposals to fast-track an independent Scotland's EU membership. The Scottish Government insisted the remarks did not apply to Scotland.
The SNP's White Paper on independence argues that in the event of a Yes vote in next year's referendum Scotland would become a full EU member seamlessly, under Article 48 of the Treaty on European Union.
Joining the EU is usually done under a process - sometimes lengthy - detailed in Article 49 of the union's key treaty.
Mr van Rompuy said: "If a part of the territory of a member state ceases to be a part of that state because that territory becomes a new independent state, the treaties will no longer apply to that territory.
"In other words, a new independent state would, by the fact of its independence, become a third country with respect to the Union and the treaties would, from the day of its independence, not apply any more on its territory."
He said any European state could apply to join the EU "according to the known accession procedures" set out in Article 49.
Though asked about Catalonia, he said his remarks were based on general principles. He said he was confident Spain would stay united.
Scottish Labour's external affairs spokeswoman Patricia Ferguson said: "Alex Salmond's position on an independent Scotland's membership of the European Union is no longer tenable.
"The most senior officials in the European Union are contradicting his assertions yet he just sticks his fingers in his ears."
Scots Tory leader Ruth Davidson said: "An independent Scotland wouldn't call the shots in negotiating entry to the EU, nor would it get any special treatment.
"It would join the back of the same queue as every other country. Mr Van Rompuy blows away Alex Salmond's assertion in the White Paper that an Independent Scotland could use a special means of entry."
Mr Van Rompuy's comments echo those of European Commission President Jose Manuel Barroso, who said a newly independent state would be treated as a "third country".
Efforts to establish beyond doubt if the Scottish Government's fast-track plan for EU membership would be acceptable to Brussels have reached stalemate.
The Scottish Government has refused to approach Brussels for clarification after the EU said it could only offer a formal legal opinion to the UK Government as member state.
The UK Government has rejected Mr Salmond's call for a joint approach, saying it cannot pre-negotiate Scottish independence. Officials have, however, described the Scottish Government's plan as a "novel" interpretation of EU law.
A Scottish Government spokesman said: "These comments are clearly and explicitly about the situation in Spain and Catalonia - not about Scotland, where the constitutional circumstances are very different, as enshrined in the Edinburgh Agreement.
"It is clear is that Scotland can negotiate the specific terms of independent membership of the European Union from within the EU, in the 18-month period between a vote for independence and independence day itself."
He said the position was backed by international experts and endorsed by the European Commission in correspondence.