The Prime Minister accused Brussels bosses of hardening their stance on Brexit just days after her dramatic Downing Street warning to them to end threats designed to influence the outcome of the June 8 General Election.
Writing in the Sun On Sunday newspaper, Mrs May said she needed the strongest possible mandate on June 8 to strengthen her hand in Brexit negotiations.
She sought to put Mr Juncker right by pointing to the importance of English as the global language of business.
Despite aides' assurance he was joking, Mr Juncker ruffled feathers in the UK with his comment, at an EU conference in Italy, that "slowly but surely, English is losing importance in Europe".
The remark came shortly amid a furious row over the leaking of details of a private dinner between the Commission boss and Mrs May to the German press, which some suspect came from Mr Juncker or his chief of staff Martin Selmayr.
German magazine Der Spiegel reported that an angry Chancellor Angela Merkel was "sour" with Mr Juncker over the leak, which she felt had "heated up" the situation in an unhelpful way.
Writing days after her emphatic victory in local elections in England, Scotland and Wales, Mrs May warned that Thursday's polls did nothing to alter "the fundamental point at the heart of the General Election campaign".
"Local council elections do not matter to bureaucrats in Brussels," she said.
"In their minds, and in those of other politicians in Europe, they do nothing to strengthen my hand for the Brexit negotiations to come.
"Indeed, even as results were rolling in on Friday, those same politicians were lining up at a conference in Italy to harden their stance ahead of the talks.
"They even took the opportunity to dismiss the importance of the English language, the language of international business that is spoken by millions of people around the world.
"That is why I am not taking anything for granted, and why I will continue to do everything I can to earn your support over the next 32 days."