The men, both in their 30s, may have been traveling the wrong way when they collided with the 18 wheeler.
It happened at Jackson Avenue and Queens Plaza South in Long Island City around 1:45 a.m. Friday.
Neither man appears to have been wearing a helmet.
One was pronounced dead at the scene, the second later died at NewYork-Presbyterian Hospital Queens.
The driver of the tractor trailer was not injured.
The incident is under investigation by the NYPD's Force Investigation Division.
Investigators are looking into whether the two men were actively fleeing police at the time of the crash.
Jackson Avenue at the Ed Koch/59th Street Bridge was closed as the police investigation continued.
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