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Early vat bacterial biofilms developed spontaneously through contact with whey have been characterized on seven wood types (Castanea sativa Miller, Cedrus libani, A. Rich., Prunus avium L., Fraxinus ornus L., Juglans regia L., Pinus nigra J.F. Arnold and Populus nigra L.). The present study aimed to evaluate the influence of these biofilms on the microbiological, chemical, physical and sensory characteristics of PDO Vastedda della valle del Belìce (VdB) cheese, processed traditionally from raw ewe's milk using wooden tools. To this purpose, the experimental cheeses after 15 d of refrigerated storage were examined. Lactic acid bacteria (LAB) populations dominated the microbial community of all samples. The species more frequently identified were Lactococcus lactis among starter LAB and Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum and Pediococcus pentosaceus among non starter LAB. Culture-independent analysis of microbiota diversity was performed by MiSeq Illumina that identified Streptococcus as major group followed by members of Enterobacteriaceae family, Lactococcus and Lactobacillus. Generally, the seven tree species did not negatively affect the physicochemical composition of VdB cheeses. Chestnut (both Sicilian and Calabrian) vats produced cheeses with significant lower hue angle (a*/b*) than other wood types. Among chemical parameters, significant variations were registered for a w , primary and secondary lipid oxidation state (significantly lower for the VdB cheeses produced with poplar wood), and volatile organic compounds (VOCs). The significant differences detected among the VOCs emitted from cheeses were not perceived by the panelists who recognized all cheeses from the different trials as similar. This study confirmed the suitability of cedar, cherry, ash, walnut, black pine and poplar as alternative woods to chestnut for the production of the wooden vats employed in cheese making for the Sicilian traditional dairy productions.