Antonella Viviani, 54, and her husband Emidio Diomede, 60, died at the scene after the bridge, which was undergoing maintenance work, gave way onto the A14 highway near Ancona on the eastern, Adriatic coast of the country.
A criminal investigation for manslaughter has been opened and the infrastructure ministry announced its own probe and sent inspectors to the scene.
The incident follows a similar one on October 28, when Claudio Bertini, 68, was killed when his car was crushed by a collapsing bridge over the SS36 dual carriageway between Milan and Lecco.
That incident was blamed on bureaucratic bungling which led to a fatal delay in the bridge being closed after it was reported to be showing significant cracks.
It came down under the weight of an articulated lorry that had been given special permission to carry an exceptionally heavy load and should have been diverted to a different road.
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The exact circumstances of Thursday's accident were unclear, but the two deaths triggered a debate about the state of Italy's infrastructure.
It also prompted questions about whether sufficient checks had been made on bridges and tunnels after three major earthquakes in the second half of last year.
The latest accident occured on the coast of the Marche region, whose mountainous interior was close to the epicentres of the quakes.
"More casualties that could have been avoided, more interventions post rather than pre, a complete lack of global vision on the security of buildings and infrastructure in Marche," said Serenella Fucksia, a senator for the opposition Five Star Movement.
Aside from the earthquake issue, Italy's infrastructure generally is showing the effects of economic stagnation with the country's output now roughly the same as it was two decades ago.
