More than 22,000 Pitbull fans were counted wearing the caps ahead of his British Summer Time headline set in Hyde Park.
The sell-out concert also became the annual gig's highest attended, with a crowd of 69,999.
Speaking before his certificate was presented, Pitbull said: "Record breaking, record making, history in the making.
"Thank you London, thank you to the fans, thank you Hyde Park, thank you to the Bald-es."
The artist, real name Armando Christian Perez, was presented with the framed Guinness World Record certificate by BBC Radio 1 presenter Greg James who came up with the idea earlier this year and texted the singer on air.
Pitbull set the record as the first person to attempt the feat under official adjudication, meaning there was no previous record to beat.
His fans, known as Bald-es, rushed to the main stage in their bald caps in order to be counted as temperatures were forecast to hit 31C in the capital.
Many sported the singer's signature black aviators, with stick-on black goatees, juggling pints of drink as they carried inflatable globes.
It comes after fans began a trend of attending Pitbull's concerts dressing up as him, sporting suits, aviator glasses and bald caps.
The trend has gone viral in recent years, with videos of audience-packed arenas full of bald cap wearing fans singing his songs back at him.
Prior to Friday's gig, the singer and festival organisers had appealed for volunteers to count bald caps in the crowd.
Supermarket chain Aldi also supplied 300 caps to Mr Worldwide fans outside the London park and temporarily rebranded itself as "Baldi".
Miami-born Pitbull is best known for the songs Fireball, Timber, Time Of Our Lives, On The Floor, Give Me Everything, Feel This Moment and Don't Stop The Party.
12°C