Problems can often arise at the beginning of the second fermentation (prise de mousse) of sparkling wines, such as no start, a long lag period or slow fermentation. These problems are generally associated with yeast stress when inoculated in a base wine with high ethanol content and low pH. However, few studies focus on sulphites, which are often added to base wines to prevent malolactic fermentation, microbiological instability, and wine oxidation. This study aimed to evaluate the joint effect of ethanol and sulfur dioxide on yeasts during the second fermentation. For this purpose, yeasts (Saccharomyces cerevisiae EC1118) were subjected to ethanol, sulfur dioxide and ethanol/sulfur dioxide at the beginning of fermentation, and their vitality and viability, as well as the accumulation of intracellular reactive oxygen species and intracellular pH, were evaluated by flow cytometry. Furthermore, the expression of genes involved in sulfur transport and metabolism was determined. The results showed high mortality, ROS accumulation and intracellular pH reduction in fermentations with both ethanol and sulfur dioxide. The negative effect of ethanol, sulfur dioxide and ethanol/sulfur dioxide on yeasts was found to be dose-dependent and high in those commonly found in some base wines. Cells treated with ethanol/sulfur dioxide showed over-expression of genes involved in sulphite transport (SUL1 and SUL2), efflux pump (SSU1 and FZF1) and metabolism of sulfur amino acids (MET14). Altogether, our data indicate that ethanol and sulfur dioxide have a synergistic effect on yeasts, which may be the root cause of the problems encountered at the beginning of the second fermentation of sparkling wines, and should thus be seriously taken into consideration by winemakers.