Aspar Ducati rider Hector Barbera provided stubborn resistance to Rossi and, after being passed, finished just over two-seconds from the #46.
Fellow Spaniard Dani Pedrosa, returning to MotoGP action after missing the last three races due to a broken collarbone in the Le Mans accident, claimed eighth, with Monster Yamaha Tech 3's Coin Edwards finishing just ahead of Hayden in ninth.
Pramac Ducati's Loris Capirossi missed his home event after being injured in qualifying for last weekend's Assen round. Capirossi will be replaced by Sylvain Guintoli for the next two events.
The Mugello circuit was resurfaced for 2011.
A test session is available at Mugello on Monday, although it now looks as though no factory 1000cc bikes will be present. The German Grand Prix at the Sachsenring takes place in two weeks' time.
Italian Grand Prix
14. de Puniet