Current global prospects of increasing ethanol production face the problem of one of the highest consumptions of fresh water and the generation of large volumes of effluents. This study evaluated the reuse of effluent from the anaerobic digestion of vinasse as a replacement of freshwater in ethanol production. This process combines the use of vinasse and anaerobic digestion effluent to produce biogas energies. The effect of reusing 0, 30, 60, and 100% effluent to dilute sugarcane molasses to produce ethanol was evaluated. The viability and vitality of the cells of the yeast were not altered by using the effluent. The efficiency of ethanol production with effluent (0, 30, 60, and 100%) was 43.3, 42.3, 39.2, 38.4 g/L, respectively. Also, we tested reusing 100% effluent without treatment for dilution of sucrose. This assay gave the highest ethanol production (80.6 g/L). The difference in ethanol production might be due to a wide range of buffering capacity of the combination of molasses and effluent, which moderately affected the performance of Saccharomyces cerevisiae. Nonetheless, additional research is required to determine a causal relationship between the buffer capacity of the molasses/effluent mixture and ethanol production.