A new approach for the detection and enumeration of Streptococcus macedonicus in cheese was developed. The method which is based on a first screening of cheeses by a PCR assay specific for S. macedonicus followed by plating positive samples on a differential medium (SM medium) was applied to 51 samples derived from PDO and traditional Italian cheeses. Streptococcus macedonicus was found in 16 of the 51 samples examined in the present work. With the exclusion of an Asiago cheese sample in which very high numbers of S. macedonicus (7.13 log CFU g−1) were found, the counts of S. macedonicus in SM medium ranged from 2.48 to 4.70 log CFU g−1. In the same cheeses, total streptococci enumerated onto M17 agar were found at higher concentrations with values up to 7.88 log CFU g−1. The system developed was particularly useful for the differential count of S. macedonicus in cheese and allowed to evaluate the occurrence of this species within the complex microbial lactic acid bacteria (LAB) population, which is typical of traditional cheeses. Results showed that in the examined cheeses S. macedonicus cannot be considered as a dominant LAB species.