The prime minister and wife Carrie were watching from the royal box when the comedian appeared to make light of the Downing Street scandal.
Mack told the crowd of tens of thousands: “Finally we can say the words ‘party’ and ‘gate’ and it’s a positive.”
The joke drew cheers and laughs from the crowd attending the concert in front of Buckingham Palace to mark the Queen's 70 years on the throne.
Mr Johnson and his wife were sat in the rows behind the Duke and Duchess of Cambridge, the Prince of Wales, the Duchess of Cornwall and the Princess Royal.
Mack’s partygate joke came only a day after Mr Johnson was booed by some sections of a crowd gathered outside St Paul’s Cathedral.
The PM, following his arrival alongside his wife at a National Service of Thanksgiving for the Queen on Friday, was jeered by some people gathered outside the church.
Mr Johnson was met with cheers and boos from the crowd at the cathedral on Friday
It is the second time a comedian has poked fun at the PM and his government over a host of Covid rule-breaching gatherings held in No 10 and Whitehall, after Mo Gilligan told Mr Johnson 'I know you love a party' as he hosted the Brit Awards in February.
London Mayor Sadiq Khan, Culture Secretary Nadine Dorries – who defended Mr Johnson after the booing incident – and Labour leader Sir Keir Starmer were all in the PM’s vicinity when Mack’s jibe was made.
Defence Secretary Ben Wallace and Home Secretary Priti Patel were also spotted in the royal box during the concert on Saturday evening, that has so far seen performances by Queen, Craig David and Andrea Bocelli among others.
Last month, an investigation published by senior civil servant Sue Gray revealed the details of a series of Covid rule-breaking events held during strict lockdown rules.
The inquiry found that excessive drinking had taken place on some occasions, with staff getting sick, altercations taking place and red wine being spilt up the walls.
The PM was found to have attended a number of leaving dos for aides, giving speeches and joining in the drinking of alcohol, despite him telling the public to avoid meeting loved ones in a bid to stop the spread of the virus.
Mr Johnson, along with wife Carrie and Chancellor Rishi Sunak, were fined by the police for attending the Prime Minister’s surprise birthday bash in Downing Street in June 2020 when coronavirus laws prohibited different households from mixing indoors.