Aims: This study aimed to investigate the dynamics and physiological heterogeneity of Oenococcus oeni under different conditions, cell membrane fluidity and permeability variations, and assessment of changes in cell surface charging rates. Methods and Results: Flow cytometry, membrane fatty acid analysis and capillary electrophoresis were performed to study ethanol-induced variations. Different physiological states were assessed, revealing cell subpopulations able to adapt and withstand to environmental stress, in order to recover their functionality. Moreover, total results demonstrated changes in cell surface and membrane fatty acid redistribution with a saturation degree and an unsaturated/saturated fatty acid ratio fairly steady in control and in different ethanol stresses. Conclusions: This study revealed a great variability among O. oeni strains and the importance to investigate the mechanisms by a multiparametric approach based on the structural and physiological bacterial adjustments in different stresses tolerance. Significance and Impact of the Study: Intermediate physiological state assessment in O. oeni with recovery possibility could be an important criterion for potential starter culture application. The flow cytometry application with changes in monitoring membrane fatty acid composition and in surface charging rates allowed the characterization of sorted subpopulations that may contribute to further understanding of diversity and heterogeneity in physiology of bacterial populations.