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Abstract: Fermentability and contamination level of wine can be assessed through the detection of viable fermentation-related and spoilage-related microorganisms. Ethidium monoazide in combination with quantitative PCR (EMA-qPCR) has been considered as a promising method to enumerate viable cells. Milling for 80 s by Ø 500-μm glass beads is demonstrated to be optimal for DNA extraction from yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) in wine to be used as a template for PCR. EMA-qPCR results from experiments using DNA extracted by this method correlate well with the results of a plating assay (R 2 > 0.99), and a PCR efficiency between 96% and 105% was obtained. Moreover, for all of these microorganisms, EMA treatment of pure cultures at a low concentration (10 μg/mL) for 20 min photoactivation resulted in effective differentiation between viable and non-viable cells and had no effect on viable cells. Due to sublethal injury to some cells, underestimation of cell counts was found in most of the wine samples tested using the EMA-qPCR method, and a 40-min incubation in recovery medium could completely offset this error. Our results suggest an optimal glass-bead DNA extraction method and EMA treatment suitable for all of the main microorganisms in wine. The EMA-qPCR method was successfully applied to quantify yeasts, Saccharomyces cerevisiae (S. cerevisiae), LAB, non-Oenococcus oeni LAB (non-O. oeni LAB) and AAB in wine samples. [Copyright &y& Elsevier]