The microbial population of raw milk plays a crucial role in the development of distinctive traits of raw-milk cheeses particularly appreciated by consumers. It was previously demonstrated that the microbial population of raw milk is modified by a high-speed centrifugation (also called bactofugation) conducted at 39 °C. The aim of the present study was to evaluate the effects of this process, performed once or twice, on the microbial, compositional, biochemical, and sensory characteristics of the derived hard cheeses. Experimental and control cheesemaking were conducted in parallel at a cheese factory during a 13-month period. Cheeses were analysed after 9, 15 and 20 months of ripening for microbial count, composition, proteolysis extent, volatile compounds, and sensory profile. Results evidenced that experimental cheeses were characterized by lower numbers of viable lactobacilli respect to control. Experimental cheeses also showed differences in the progress of primary and secondary proteolysis which, in turn, caused different patterns of free amino acids at all ripening times. Experimental cheeses had significantly lower content of esters and were differentiated from control for some traits by assessors. In conclusion, use of high-speed centrifugation of milk shall be discouraged if characteristic traits of raw-milk cheeses, particularly PDO cheeses, want to be retained.