The departure of the MSC Grandiosa from the northwestern port city represents a high-stakes test for the global cruise sector.
The crisis forced operators to ground their ships and also led to accusations that they botched the handling of the epidemic in its early stages.
Cruise lines are hoping that tighter protocols will allow them to control the still-lingering threat of coronavirus aboard its ships while still offering travellers a cruise experience that does not disappoint.
Any one testing positive, or with a fever, or having other COVID-19 symptoms, was denied boarding, the company said.
Guests must wear face masks in elevators and other areas where social distancing is not possible. The crew spent time in quarantine before the start of the cruise.
Earlier this month, the Italian government gave its approval for cruise ships to once again depart from Italy's ports, but limited cruise ships to sailing with 70 per cent capacity.
The cruise company declined to say how many passengers were sailing on this cruise. Among the port calls for the Grandiosa, MSC's flagship, are Naples, Palermo, Sicily and Valletta, Malta.
Malta is one of four Mediterranean countries that Italy now requires travellers arriving from to have COVID-19 tests.
