The LBC pundit said the European Union's strongest pillars, freedom of movement and human rights, would be one of the "surest ways" to guard against any rise similar to that of the Nazis in the 1930 and 40s.
He took aim at the claims of pro-Brexit campaigner Johnson, who yesterday drew parallels between the political union and Hitler, saying the German dictator had failed at unifying Europe but that the EU was "an attempt to do this by different methods".
Responding, O'Brien questioned: "What are the big things we're told the European Union is bad for? Freedom of movement is supposed to be our enemy and...human rights is quite big actually.
"Hitler's record on freedom of movement and human rights, I'm going to go out on a limb here because I know how dangerous it is to cite Hitler in the context of modern politics. I'm going to go out on a limb here and cautiously suggest... Hitler's record on freedom of movement and human rights was not great."
"So the people telling us the EU is evil are the same people that are opposed to freedom of movement and human rights legislation.
"And they're the same people today that are cheering Boris Johnson for comparing an organisation that they deride for its commitment to freedom of movement and human rights legislation - for comparing it to Hitler.
"That truly is evidence that we're down the rabbit hole - and I don't know if we're ever going to find our way out."
O'Brien continued: "You can’t help thinking that if Winston Churchill were alive today, he would probably use freedom of movement and human rights as the rationale behind, well, certainly, behind his support for the convention on Human rights.
"But the surest way to guard against the horrors visited upon continental Europe by the Nazis is surely to bring the countries closer again isn’t it?"
Johnson - seen as the de facto leader and highest profile member of the Leave campaign - said the past 2,000 years of European history had been dominated by doomed attempts to unify the continent under a single government to recreate the “golden age” of the Romans.
“Napoleon, Hitler, various people tried this out, and it ends tragically. The EU is an attempt to do this by different methods,” he said in an interview with The Sunday Telegraph.
“But fundamentally what is lacking is the eternal problem, which is that there is no underlying loyalty to the idea of Europe.
“There is no single authority that anybody respects or understands. That is causing this massive democratic void.”
His comments came as Nigel Farage backed Johnson to succeed David Cameron if there is a vote to leave the EU in the referendum on June 23.