Biofuels are an important alternative, renewable source of energy in the face of the ongoing depletion of fossil fuels. Cheese whey is a dairy industry waste characterized by high lactose concentration, which represents a significant environmental problem. Bio-ethanol production by cheese whey could be an effective nonvegetable source for renewable energy production. Here, we report the isolation of a mixed microbial population, able to produce ethanol as main fermentation product from fermenting whey. The microbial consortium has been used to perform a batch fermentation of crude whey in both anoxic and hypoxic conditions. Maximum ethanol concentrations achieved in this study was obtained using the mixed culture in hypoxic conditions, grown at pH 4 and 30 °C, with ethanol production yield of 60 g/L. Our research has pointed out an alternative way to both dispose and valorize cheese whey, a dairy by-product that could cause water pollution and harm to the environment if not properly treated.