The Commission president has faced a growing continental backlash since he declared last month that Scottish membership would be "difficult if not impossible".
Now, in a morale-booster for the Yes campaign, Mr Barroso's assertions have come under direct fire from former Czech president Vaclav Klaus and Joelle Garriaud-Maylam, a senior French senator specialising in foreign policy.
Most Brussels watchers believe Mr Barroso's words, on the BBC's Andrew Marr Show, were directed as much at Catalonia - which will hold an unsanctioned vote on independence from Spain later this year - as they were at Scotland.
However, Mr Klaus, speaking at a conference on secession movements in Austria, made it clear he expected Scotland and Catalonia, if they were independent, to stay in the EU.
Answering a question from Professor Charlie Jeffery, of Edinburgh University, Mr Klaus said: "It is arrogant of the EU to say Scotland and Catalonia will not be members."
His words came after another prominent conservative, Mme Garriaud-Maylam, effectively accused the UK and Spain of being behind Mr Barroso's intervention.
Speaking in the French Senate, Mme Garriaud-Maylam said: "The threats formulated by Mr Barroso are inappropriate and the result of Spanish and English pressure. London is increasingly worried. They (the threats) are not credible. If Scotland votes for independence, it will stay in the European Union. It would be in England's interest."
The French Conservative is secretary of the Senate's Foreign Affairs, Defence And Armed Forces commission and was delivering a special statement to parliament on Scotland and the EU.
She stressed that states with their own separatist concerns, such as Spain, Belgium and Italy, would "not seek to facilitate accession, as well demonstrated by Mr Barroso's recent comments".
She added: "Large member states will probably seek to maintain the status quo.
"However, the realism of these declarations should be questioned, given the practical consequences of suspending EU co-operation, which, in this case, has existed for more than 40 years and which Scotland wishes to retain.
"It seems reasonable to think most member states will align their position with London, which has already signalled it would respect the results of the referendum."
Mme Garriaud-Maylam's views tally with those of Scottish expert Professor Michael Keating, who said: "I don't see any political will to exclude Scotland from the EU. It is not in the interests of any of the member states, including the UK and Spain."
EU federalists, such as Commission Vice-President Viviane Reding, another Conservative, have made it clear they do not wish the bloc to "lose territory" because of secession movements.
A spokesman for pro-UK campaign Better Together said the "fundamental question is how would we get back in and on what terms".
He added: "If we do manage to get the approval for our application from all of the EU countries, then there is no doubt that we would have lost our position, through being part of the UK, as one of the most influential countries.
"Only the SNP could possibly think that putting ourselves through all of this just to make ourselves less influential."