Nationalists believe an independent Scotland would have no problem remaining in the European Union.
Barroso has said any country winning independence from a current EU member state would have to renegotiate its membership.
“In the hypothetical event of a secession of a part of an EU member state, the solution would have to be found and negotiated within the international legal order,” he said in recent remarks.
Unionist politicians jumped on the statement, which they said meant an independent Scotland risked losing EU membership.
David Martin, a Labour MEP and the party's spokesman on constitutional affairs, said membership was not automatic.
"An independent Scotland's membership of the EU is far from clear, and there is no precedent to suggest membership would be automatic," Martin said.
“President Barroso’s recent comment reflects what the European Commission has been saying for a long time," he said, adding that the situation is "certainly not as clear-cut as the SNP would like to believe."
Martin added the European Commission, the European Parliament and all member states would have to agree to Scotland becoming a new member.
A Scottish flag in Brussels
But the Scottish Government have said there is nothing in Barroso’s response that suggests Scotland will not retain its place in the EU.
SNP MEP Alyn Smith said unionist parties were scare-mongering.
“The president has made it clear he is not going to get involved in these discussions because this is a matter for the people of Scotland. It is utterly inconceivable this could be any other way," Smith said. “Membership is a formality and the people of Scotland will decide.”
A European Commission spokeswoman declined to be drawn into the issue of Scottish independence.
She said: “At the present time, the Commission is not able to express any view on the specific issue raised given that the terms and result of any future referendum are unknown, as is the nature of the possible future relationships between the parties concerned and between those parties and European Union partners.
“The Commission would recall in this context that the terms of any European Union Treaty are decided by the member states of the European Union.”