The 35-year-old comic, a Berlin native, is known for broad humor about gender stereotypes. On Saturday he hit familiar notes with jokes about shopping-addicted women, macho men and beer.
The arena had been sold out for months for the show, the final stop on Barth's two-and-a-half-year tour “Men are primitive, but happy.” The solo shows sold 1.7 million tickets.
Before the show, Barth said American comedian Jerry Seinfeld was perplexed when he heard of the world record attempt.
“He couldn't imagine that a single man would perform in front of so many people,” Barth said.
The crowd at the Berlin show was more than four times larger than the previous world record-holder, a 15,900-person show by American comedian Chris Rock in London, a Guinness World Records spokesman said.
German rock group Silbermond opened for Barth as a surprise guest.
Singer Stephanie Kloss told the crowd the group had lost a bet to Barth.
“This is what happens when you have a beer with Mario Barth and tell him, ‘You'll never fill up the Olympic Stadium,'” Kloss said.