The European Council president seemingly backed Mr Johnson’s opponents in the upcoming general election by saying the UK will become "an outsider, a second-rate player" after Britain leaves the EU.
He added that a friend who suggested it is "the real end of the British Empire" is probably right.
In remarks that are likely to infuriate Brexiteers, Mr Tusk said: "The UK election takes place in one month. Can things still be turned around? Hannah Arendt taught that things become irreversible only when people start to think so.
"So the only words that come to my mind today are simply: Don't give up. In this match, we had added time, we are already in extra time, perhaps it will even go to penalties?”
Speaking at the opening ceremony of the 2019/2020 academic year at the College of Europe, Mr Tusk added: "I have heard repeatedly from Brexiteers that they wanted to leave the European Union to make the United Kingdom global again, believing that only alone, it can truly be great.
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"You could hear in these voices a longing for the Empire. But the reality is exactly the opposite. Only as part of a united Europe can the UK play a global role, only together can we confront, without any complexes, the greatest powers of this world.
"In fact, I can say the same about Germany or France. And the world knows it. I have heard the same in India, New Zealand, Australia, Canada and South Africa; that after its departure, the UK will become an outsider, a second-rate player, while the main battlefield will be occupied by China, the United States and the European Union.
"'Why are they doing this?' - I was asked this regretful question everywhere I went. One of my English friends is probably right when he says with melancholy that Brexit is the real end of the British Empire.”
Referencing Mr Bercow, who described Brexit as "the biggest foreign policy mistake in the post-war period", Mr Tusk said: "Finally, if you allow me, I want to tell you something I wouldn't have dared to say a few months ago, as I could be fired for being too frank.
"And today, it is simply too late to impeach Donald, at least the European one. In fact, I envy John Bercow, that he can finally, honestly say what he thinks about Brexit.”
Mr Johnson is attempting to secure a majority in the December election that will allow him to take Britain out of the EU by the end of January.
However, Labour plan to renegotiate the deal with the EU and put it to a second referendum in 2020 if they win next month, while the Lib Dems want to revoke Brexit altogether.
Mr Tusk’s comments come just weeks before he is set to stand down from his role as EC president.