Artisanal cheeses often rely on indigenous microorganisms present in raw ingredients, equipment, and the environment. Biofilms on wooden tools utilized in cheese-making activities act as reservoirs of lactic acid bacteria with techno-functional properties that contribute to fermentation processes and, ultimately, the characteristics of the final product. However, the microbial succession across the different fermentation stages remains poorly defined. A combined culture-dependent and -independent approach was applied to define the microbial succession in two artisanal stretched-curd cheeses, Provola dei Nebrodi PDO and Ragusano PDO. Through this approach, the microbiota associated with the wooden acidification-table biofilms, pre- and post-acidification curd,  and final cheese were evaluated. The culture-based analysis, combined with sequencing of 16S rRNA for bacteria and ITS for lactobacilli and fungi, highlighted the dominance of Lactobacillus species. Therefore, a qPCR-based system was designed for the molecular “tracking” of such key taxa to follow their progression during the production process. The wooden biofilms were dominated by Lactobacillus (~90% relative abundance), primarily Lb. kefiranofaciens, alongside Lb. gasseri, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Lb. reuteri. Pre-acidification curds were dominated by Streptococcus (~75–79%), with Lactobacillus accounting for ~13–15%. Following acidification, the abundance of Lactobacillus was ~50–60%, which is consistent with the selection of species promoted by the wooden biofilms. This study provides an integrated, validated framework for tracking microbial dynamics in artisanal fermentation processes and provides targeted monitoring tools to support quality assurance and authenticity in traditional dairy systems.