Chlorothalonil is a broad-spectrum fungicide largely used for the control of several diseases of grapevines. With a moderate persistence in plants, soil and, water, it can be carried to grape musts, particularly when applied to control grape rot diseases. This work aimed to determine the effect of chlorothalonil on Saccharomyces cerevisiae under fermentative conditions using a flow cytometry approach. Yeasts were cultivated in synthetic must with different concentrations of chlorothalonil (0 to 60 μM) and evaluated for culture-ability, membrane integrity, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential, metacaspase activity, ATP, nonprotein SH and, SH-proteins. The results confirmed the oxidation of nonprotein SH, including glutathione, and the binding of the fungicide with sulfhydryl proteins, which led to changes in the cell and mitochondrial membranes that result in the necrotic death of part of the yeast population, and a reduction in metabolic activity. Moreover, the reduction in glutathione-SH concentration was responsible for the increase in ROS which in turn triggers metacaspase-dependent apoptotic cell death. Cells that escape death adapt and began to grow and ferment after a dose-dependent lag-phase period, exhibiting an almost normal fermentative behavior thereafter. Moreover, was observed unexpected protection of chlorothalonil sub-dosages on yeast cell membrane integrity during alcoholic fermentation. This study contributed insights into how chlorothalonil leads the non-target organism S. cerevisiae to cell death and explores the effect of the fungicide during alcoholic fermentation.