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Caciocavallo Palermitano” is a raw cows’ milk cheese produced within Palermo province (Sicily), processed according to “pasta-filata” cheese technology without starter addition. Production flow-sheet includes a 15-minute milk bulk resting in “tina”, the traditional wooden vat used for cheese-making. This study was aimed to evaluate the influence of traditional equipment on the characteristics of cheese; at this stage, it was forwarded to the characterization of the microbiological populations of “Caciocavallo Palermitano” during each step of manufacturing. Two milk bulks (A and B), from different farming systems, were compared before and after resting in tina. After delivery at dairy factory, total microbial counts (TMC) differed of about 2 Log cycles (4.33 and 6.14 CFU/mL for milk bulk A and B, respectively). Mesophilic lactic acid bacteria (LAB) were counted at about 103 CFU/mL, both rods and cocci, for milk bulk A, while ca. 105 CFU/mL for milk bulk B. Similar differences, in terms of magnitude order, were registered for termophilic LAB, even though LAB rods were approximately 1 Log cycle lower than LAB cocci (102 and 104 CFU/mL for bulk milk A and B, respectively). Milk bulks were also analysed for psycotrophic bacteria, total colyforms, enterococci, pseudomonads, coagulase positive staphylococci, clostridia and yeasts. Interestingly, after resting in tina, milk bulk A was characterized by 105 CFU/mL of TMC and mesophilic and thermophilic LAB. Similar counts were detected for milk bulk B. Thus, tina colonizing LAB determined the increment of milk LAB population. Cheese production was microbiologically analysed from curdling to stretching, at which the dominance of thermophilic rod and cocci LAB was registered for both milk bulk productions. LAB isolated at the different stages are being identified and typed, in order to verify the persistence of thermophilic LAB that colonize tina, and characterized for their technological potential and contribution to “Caciocavallo Palermitano” typicality.