Jean-Claude Juncker's chief of staff, Martin Selmayr, later launched his own attack by calling the prospect of Mr Johnson as Prime Minister alongside other prospective world leaders a "horror scenario".
Tweeting from the G7 summit, he wrote: “#G7 2017 with Trump, Le Pen, Boris Johnson, Beppe Grillo? A horror scenario that shows well why it is worth fighting populism. #withJuncker."
Mr Juncker had been asked about the Conservative MP’s comparison of efforts to unify Europe with the actions of Napoleon and Hitler at the G7 summit in Japan.
"I'm reading in the papers that Boris Johnson spent part of his life in Brussels,” Mr Juncker said. "It's time for him to come back to Brussels, in order to check in Brussels if everything he's telling British people is in line with reality.
"I don't think so, so he would be welcome in Brussels at any time."
Mr Johnson hit back in an interview with Sky News, saying his arguments "were in line with reality".
"If we vote to remain, which I sincerely hope we don't on 23 June, they will go on with measures to take us further into a European superstate...it will tend towards the creation of a United States of Europe," he said.
Mr Johnson, now leading the campaign for a Brexit, spent several years in Brussels as EU correspondent for The Daily Telegraph before entering politics, with his articles noted for their Euroscepticism.
In an interview with his former employer earlier this month, he said: "The history of the past couple of thousand years has been broadly repeated attempts by various people or institutions... to rediscover the lost childhood of Europe, this golden age of peace and prosperity under the Romans, by trying to unify it. Napoleon, Hitler, various people tried this out, and it ends tragically.
“The EU is an attempt to do this by different methods.”
When asked whether the European institutions would be able to work with Mr Johnson if he suceeded David Cameron as leader of the Conservative Party and became Prime Minister, Mr Juncker quipped: “The atmosphere of our talks would be better if Britain is staying in the European Union.”
He indicated he has little doubt over the result of the referendum on 23 June, telling reporters: “Britain will remain as a member of the European Union.”
Donald Tusk, president of the European Council, said people must respect democratic decisions and their “political consequences”.
“I think it's quite normal to have normal relations with politicians and at the same time to have your own opinion about their opinion,” he added.
On the day when the Office for National Statistics is due to publish its final migration figures before the referendum, Mr Tusk acknowledged that the movement of workers from EU states to the UK was one of the key drivers behind British Euroscepticism.
But he insisted nations that subscribe to the fundamental principles of the EU – such as freedom of movement – must be ready to accept “negative results” from the policy.
“We are absolutely aware what are the main reasons of the referendum, and for sure internal migration from the EU states is one of the main reasons of Euroscepticism in the UK,” Mr Tusk said.
“But we have to choose. If we prefer free movement, if we prefer the whole values of the EU, we have to accept also some negative results."
Additional reporting by PA
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