We applied flow cytometry (FCM) to monitor microbial dynamics during grape must fermentation at the winery scale. Experiments were performed on Pinot Noir grapes using three distinct winemaking protocols: inoculation with active dried yeast, Pied-de-Cuve, and spontaneous fermentation. FCM enabled the assessment of yeast viability and metabolic activity, as well as the detection and monitoring of viable bacterial populations during alcoholic fermentation. Amplicon-based DNA sequencing was performed to characterize the associated microbial communities and evaluate protocol-specific effects. Trends identified by amplicon sequencing were partially mirrored by patterns observed in unsupervised FCM analysis. Overall, our results indicate that FCM is a practical tool for monitoring microbial dynamics during fermentation, providing near–real-time information that can support monitoring strategies and risk management in winemaking