John Paul McManus and John Magnier, whose holding company was already the club's biggest single shareholder with an 11.4% stake, now owned 23.15%, Manchester United told the London Stock Exchange on Tuesday.
The bulk of the extra shares were bought from British satellite broadcaster BSkyB, which announced earlier in the day it had sold its 9.9% stake for about £62 million ($103 million).
Mystery surrounded the intentions of McManus and Magnier, a pair of super-rich racehorse owners and enthusiasts. They have long been friends with Manchester United team manager Alex Ferguson, a fellow racing fan who is one of the most influential figures at the club.
The buy-up comes less than a week after Manchester United revealed that US tycoon Malcolm Glazer had doubled his holding in the club to 5.9% in less than a week.
Last month, a series of media reports said another US businessman Philip Anschutz was poised to make an offer for the club.
News of the latest share buy-up sent United shares - which have already climbed strongly in recent weeks - up a further 4.1% to 247 pence by late afternoon trade.
The club is widely regarded as the
world's biggest and richest
It remained by no means clear what the Irish duo, who own their shares through a British Virgin Islands-registered investment firm Cubic Expression, planned to do with the increased stake.
"It's very difficult to know what their intentions are, and I'm sure the club will want to speak to them to find out," said Lee Darbyshire, editor of London-based Soccer Investor magazine, which covers the world of football finance.
"Part of me thinks that they could just be increasing their stake in case someone else attempts to make a bid," he said. "But it's very difficult to say."
Barclays Private Clients analyst Henk Potts said it was possible the pair had ideas of their own.
"It makes no sense for them to have such a large holding in a company like Manchester United and not be in overall control of the club," he said.
Takeover rumours have swirled around a series of English sides since Russian oil billionaire Roman Abramovich snapped up Premiership side Chelsea over the summer.
Manchester United are seen as a particularly attractive proposition for predators owing to the combination of their success on the pitch and huge commercial revenues from sponsorship and merchandise sales.
Last week, United - generally reckoned to be the richest as well as possibly the most famous football club in the world - announced a 22% rise in annual profits to £39.3 million.