Other acts confirmed for the four-day event in Manchester, Tennessee include Al Green, Snoop Dogg, Nine Inch Nails, David Byrne and Elvis Costello.
Paul Oakenfold, Erykah Badu, The Mars Volta, TV On The Radio, Yeah Yeah Yeahs and MGMT are also on the bill.
Bonnaroo, which takes place on June 11, 12, 13 and 14, will be Springsteen's only North American festival date. It is the first confirmed Beastie Boys live show of 2009.
"We are looking forward to bringing heat and fire inside of your festival grounds," the rap trio told Billboard. "We [the band known as Beastie Boys] are like a three-headed dragon with hot molten metal spewing forth from every head. Ready yourselves. Stand fast. Saturn's second moon is on the rise. We approach you now from the northeast at the speed of lightning. There will be no respite from our wrath."
Tickets go on sale on Saturday.