The trio entertained 180 passengers at 35,000 feet aboard a British Airways plane for the record-breaking 'Smile High' event, which was organised by the airline as part of its partnership with Comic Relief.
The funnymen performed for a total of 45 minutes on the two-and-a-half-hour flight, which took them into Norwegian airspace before returning to London's Heathrow airport.
Metro reports that 150 of the crowd were competition winners, who raised nearly £100,000 for the charity in entry fees.
Speaking after the unique gig, O'Briain admitted: "I didn't do any preparation - we were all just scribbling notes down at the start of the flight!
"We can't normally make jokes about aeroplanes though as it's a sensitive subject, so it was quite hard to think of new ones but all of our material should be on the tips of our tongues."
British Airways captain Brian Connolly added: 'It was an honour to captain this very special flight today. I'm proud to be able to say that I'm a Guinness World Records holder, and of course - member of the 'Smile High' club!"
The 'Flying Start' partnership between British Airways and Comic Relief has raised over £700,000 since its launch in June 2010.
Red Nose Day takes place on March 18.