The One-day International will be a repeat of the World Cup final contested by the two sides at the home of cricket in 1999 which the Australians won by eight wickets.
The newly-arranged fixture will provide both teams with a chance to prepare for the international Cricket council (ICC) Champions Trophy which will be held in England from September 8 to 25.
The two sides will also be taking part in a mini-tournament involving India in Amstelveen, near Amsterdam in Holland from August 21 to 30.
The Lord's fixture will be the first of two major matches on successive days at the venue.
England will face India in the final game of their three-match NatWest Challenge series on September 5.
MCC's secretary and chief executive Roger Knight said: "We are delighted to be welcoming Australia and Pakistan, two world-class teams, to Lord's in September.
"Australia will be keen to retain their reputation as the best team in the world while Pakistan will be desperate to avenge their defeat by the Australians in 1999's World Cup final at Lord's.
"We were already expecting an exciting summer with our first Twenty20 match next month, and England due to play the West Indies in July and India in September.
"For everyone at Lord's this extra fixture is set to make an outstanding summer even better."
MCC will make an announcement about ticket sales nearer the event.