The bioethanol fermentation by Saccharomyces cerevisiae is often challenged by bacterial contamination, especially lactic acid bacteria (LAB). LAB can inhibit the growth S. cerevisiae by secreting organic acids and competing for nutrients and physical space. However, the range of favorable effects attributed to LAB during bioethanol fermentation, and their associated mechanisms of regulation, are not fully understood. This study was performed to clarify the effects of Lactobacillus plantarum, an important contaminative LAB in bioethanol fermentation, on the mechanism of ethanol tolerance in S. cerevisiae. The results showed that the presence of L. plantarum increased the ethanol tolerance of S. cerevisiae by promoting or inhibiting various metabolic processes in the yeast cells: The metabolism of trehalose, ergosterol, certain amino acids, proton pumps, stress response transcriptional activators, and heat shock proteins were all promoted; amounts of intracellular monounsaturated fatty acids and the accumulation of reactive oxygen species were inhibited. Furthermore, the maintenance of the acquired higher ethanol tolerance of S. cerevisiae was dependent on the coexistence of L. plantarum. These results suggested a complex relationship existed between S. cerevisiae and the contaminating LAB that might also play a beneficial role during fermentation by promoting the ethanol tolerance of yeast. The results from this study suggested that the extent of controlling bacterial contamination on bioethanol fermentation efficiency should be given careful consideration.