Ragusano cheese is a "protected denomination of origin" cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called "tinas" were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii, and Enterococcus faecium. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected, S. thermophilus being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (S. thermophilus, L. lactis, and L. delbrueckii subsp. lactis) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system.