Jose Manuel Barroso refused to speculate on indepedence movements within specific member states during an interview on BBC Radio 4.
However, he inisisted: "To join the EU, yes, we have a procedure and it is a procedure in inter-national law. A state has to be a democracy and has to apply to become a member of the EU. All the other member states have to give their consent."
His comments followed questions about the future status of Scotland and Catalonia within the EU, should their independence movements prevail, and sparked fresh clashes between Mr Salmond's Government and opposition parties.
Scottish Labour leader Johann Lamont said Mr Barroso had raised the serious prospect of an independent Scotland having to join the euro single currency and urged the First Minister to reveal the Government's legal advice on EU membership. Mr Salmond acknowledged that an independent Scotland would have to negotiate the terms of its EU membership.
The row follows comments by a European Commission spokesman, Olivier Bailly, on Tuesday who said a new country would have to make a request for accession.
Answering a question in Strasbourg about Catalonia and Scotland he said: "If it [the seceding country] wants to apply for EU membership then this has to be done according to the treaties and the provisions regarding accession. There are two different steps. There is a secession process under international law and the request for accession to EU member state under the EU treaties."
Following an approach from Scottish Government officials he issued a clarification saying moves for independence were "first and foremost an internal matter within that member state".
Ms Lamont last night called on the Scottish Government to drop legal action aimed at preventing the disclosure of legal advice on EU membership, which has been ordered by Freedom of Information watchdogs.
She added: "Alex Salmond has found himself in a ridiculous position when he is blindly arguing that Scotland will automatically be a full EU member while senior figures in Europe are contradicting him.
"The SNP has to be transparent and honest about Scotland's future."
Scottish Conservative leader Ruth Davidson MSP said: "Alex Salmond is already spending considerable sums of public money trying to suppress whether or not he has taken legal advice on EU entry.
"Now we have the European Commission reaffirming what everyone knows – that a separate Scotland cannot simply waltz into the EU unchallenged."
Scots LibDem leader Willie Rennie said: "It looks like SNP ministers may have been misleading everyone."
Asked about Mr Barroso's intervention, Mr Salmond said: "Our position is quite clear. Yes, there will have to be negotiations. The negotiations will take place from within the context of the European Union.
"The negotiations are about the representation Scotland will have and the rest of it."
His chief political spokesman added: "Scotland would continue in EU membership. We are an integral part of the EU and will inherit the same treaty rights and obligations as the rest of the UK."
A Foreign Office spokesman said: "It is clear the Scottish Government cannot take for granted the idea Scotland would secede from the UK but automatically stay in the EU. The most likely scenario would be that Scotland would have to join the EU as a new state, requiring negotiation with other Member States.
"The Scottish Government needs to be clear on the facts of what independence actually means in practice."