In this work the effect of the milk bactofugation and natural whey culture on the microbiological and physicochemical quality of Mozzarella cheese was studied. To this aim, the microbiological, sensory and physico-chemical parameters of the Mozzarella cheese were monitored during the storage at 8°C. The bactofugation treatment did not significantly affect the growth of typical dairy microorganisms, while a significant decrease of Enterobacteriaceae in milk was found. The Mozzarella manufactured with natural whey culture and bactofugated milk showed a slower increase in the Pseudomonas spp. cell load during storage. Moreover, the absence of natural whey culture in the Mozzarella cheese manufacture caused a faster sensorial quality loss during storage (~4.5 days) respect to the product with natural starter (~6 days). However, the factor limiting the shelf life of Mozzarella cheese was the growth of Pseudomonas spp. In particular, a shelf life value of about 4.0 days was obtained for Mozzarella produced with bactofugated milk and natural whey culture versus 3.5 days for the control, with citric acidification, and samples manufactured with natural whey culture and no bactofugated milk. The compositional characteristics of the cheeses were influenced by the use of the natural starter, especially at the end of the storage period. In fact, the control cheese tended to lose soluble compounds (WSN and NaCl) faster and, increase in moisture content.