The dynamics of dominant microflora throughout the manufacture and ripening processes were evaluated in three batches of traditional Castelmagno PDO cheese. Milk, curd and cheese samples, at different stages during cheesemaking, were collected and subjected to culture-dependent and -independent analysis. Traditional plating and genetic identification of lactic acid bacteria (LAB) isolates, and PCR-DGGE analysis of V1 region of 16S rRNA gene were carried out. The collected samples were also monitored by HPLC for the presence of organic acids, sugars and ketones. LAB resulted to be the prevailing microflora in all production stages although enterococci, coagulase-negative cocci and yeasts also showed considerable viable counts probably related to the presence, in the dairy samples analysed, of free short-chain fatty acids detected by HPLC. Lactococcus lactis subsp. lactis was the species most frequently isolated during Castelmagno PDO manufacture, while Lactobacillus plantarum and Lactobacillus paracasei were isolated with the highest frequencies from ripened Castelmagno PDO cheese samples. Occasionally strains of Lactobacillus delbrueckii subsp. lactis, Lactobacillus coryniformis subsp. torquens and Lactobacillus casei were isolated. The results obtained on Castelmagno PDO microflora underlines a partial correspondence between culture-dependent method and DGGE analysis. Thus, in this study, it is highlighted once more the importance to combine molecular culture-independent approaches with classical microbiological methods for the study of complex environmental communities occurring in food matrices.