Tolerance to high temperature and ethanol is a major factor in high-temperature bio-ethanol fermentation. The inhibitory effect of exogenously added ethanol (0–100 g L−1) on the growth of the newly isolated thermotolerant Issatchenkia orientalis IPE100 was evaluated at a range of temperatures (30–45°C). A generalized Monod equation with product inhibition was used to quantify ethanol tolerance, and it correlated well with the experimental data on microbial growth inhibition of ethanol at the temperatures of 30–45°C. The maximum inhibitory concentration of ethanol for growth (Pm) and toxic power (n) at the optimal growth temperature of 42°C were estimated to be 96.7 g L−1 and 1.23, respectively. The recently isolated thermotolerant I. orientalis IPE100 shows therefore a strong potential for the development of future high-temperature bio-ethanol fermentation technologies. This study provides useful insights into our understanding of the temperature-dependent inhibitory effects of ethanol on yeast growth.