Police released video showing one of the men standing in front of the bus, who they say then punched the driver and spit in his face.
The other man forced the doors of the bus open.
Police say both men boarded and harassed the driver before fleeing the scene.
The incident happened on Wednesday, March 24, around 2:15 p.m. on a BX6 express bus in the Bronx.
The victim suffered pain to his left arm, shoulder, and neck.
MTA officials said they were outraged over the attack.
"This was an abhorrent act of violence and disrespect against a transit hero just doing his job moving New Yorkers," MTA Bus President Craig Cipriano said. "We are cooperating fully with the NYPD, join in asking for the public's help identifying perpetrators, while this further highlights the need for a stronger police presence on buses."
Anyone with information about this incident is asked to call the NYPD's Crime Stoppers Hotline at 1-800-577-TIPS (8477) or for Spanish, 1-888-57-PISTA (74782).
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