Jean-Claude Juncker has been branded an "unelected tinpot figure" after warning there will be no further renegotiations should voters back Brexit.
The European Commission president insisted "out is out" if Britain opts to leave the EU, adding David Cameron has secured the "maximum" Brussels could give.
But senior Vote Leave campaigner Boris Johnson hit out at Mr Juncker's remarks and claimed people have been "told from the horse's mouth" that hope of further changes are an "absolute illusion".
Mr Juncker's intervention came as Nato secretary general Jens Stoltenberg also played up the UK's role in boosting security, warning a fragmented Europe will "add to instability and unpredictability".
Speaking while on the campaign trail ahead of Thursday's vote, Mr Johnson said: "Who elected Jean-Claude Juncker to run anything in this way? Who put him in charge of us in this way?
"This gives the game away. If we stay in there is no prospect of any further change.
"This is it, folks. We have been told from the horse's mouth that any hope of further change is absolute illusion."
Speaking in Brussels, Mr Juncker had earlier told reporters: "The British policy makers and British voters have to know that there will be no kind of any renegotiation.
"We have concluded a deal with the Prime Minister, he got the maximum he could receive and we gave the maximum we could give.
"So there will be no kind of renegotiation, nor on the agreement we found in February, nor as far as any kind of treaty negotiations are concerned.
"Out is out."
Ukip leader Nigel Farage tweeted: "Jean-Claude Juncker directly contradicts the Prime Minister. There is no further renegotiation."
Jean-Claude Juncker directly contradicts the Prime Minister. There is no further renegotiation. https://t.co/UvpBELCHQj— Nigel Farage (@Nigel_Farage) June 22, 2016
He added: "Mr Juncker confirms this is Britain's last chance saloon. We must vote to Leave EU."
Mr. Juncker confirms this is Britain's last chance saloon. We must vote to Leave EU.— Nigel Farage (@Nigel_Farage) June 22, 2016
Mr Stoltenberg sought to praise the UK for acting as a "bridge" between the EU and Nato, plus Europe and the United States, while outlining the country's role in combating terrorism and other issues.
He told the Guardian: "I don't have a vote. It's up to the people of Britain to decide.
"What I can do is tell you what matters for Nato, and a strong UK in a strong Europe is good for the UK and it's good for Nato, because we are faced with unprecedented security challenges, with terrorism, with instability and an unpredictable security environment, and a fragmented Europe will add to instability and unpredictability."