The Doctor added a 114th victory to his career win tally with an emphatic triumph in the Grand Prix of Catalunya, despite falling as low as ninth on the opening lap.
The race weekend has been marred by the death of Moto2 rider Luis Salom, who was killed in a crash at turn 12 in Friday's free practice.
That incident led to a revised track layout to be adopted, with Movistar Yamaha duo Rossi and Lorenzo quick to fume at the changes, claiming they gave the advantage to the Repsol Hondas of Marquez and Dani Pedrosa.
But it was Rossi who claimed maximum points, holding off Marquez to seal victory - making the telling move with two laps remaining - with Pedrosa completed the podium.
Despite settling for second, Marquez is now 10 points clear of Lorenzo at the top of the standings after the defending world champion was knocked off his bike by Andrea Iannone.
Iannone, who will leave Ducati at the end of the season to allow Lorenzo to join the team, careered into the back of the Spaniard's bike, unseating both riders and handing Marquez the advantage in the title race.