Tsvangirai's Movement for Democratic Change (MDC) has joined forces with Mugabe's former finance minister Simba Makoni and the Zanu-Ndonga party ahead of elections on July 31.
The three formed a "coalition for change" behind Tsvangirai's bid for president, according to a joint statement.
Makoni, a former senior official in Mugabe's Zimbabwe African National Union - Patriotic Front (Zanu-PF), quit the party in 2008 to form his own party Mavambo-Kusile-Dawn and challenge Mugabe in the presidential race.
The third partner in the coalition is Zanu-Ndonga, a party that was allied with Mugabe during the fight for Zimbabwe’s independence, but later split.
The three partners said they will support each other in the parliamentary and local authority elections and form a "government of national unity" if they win the vote.
"The government of national unity will be based on the free will of the people as opposed to the current coalition government created after the subversion of the will of the people in the 2008 presidential election," Makoni said.
Mugabe, 89, and Tsvangirai, 61, were forced into a shaky power-sharing government four years ago after a bloody presidential run-off election which Tsvangirai boycotted citing violence against his supporters.
Some 200 opposition activists were killed in the election unrest.
The announcement of Tsvangirai's partnership with two other parties comes just days after a splinter group from his MDC signed an agreement with the powerful Zimbabwe African People's Union (ZAPU) to support each other's candidates.