Drosophila suzukii (Matsumura) (Diptera: Drosphilidae) is an important pest that causes damage to fruits of over 60 plant species. Drosophila suzukii oviposits on ripe fruit, while D. melanogaster oviposits on decaying fruit. Therefore, these species occupy separate ecological niches. To provide a better understanding of the alcohol tolerance between these 2 species and explore the relationship of ecological niche differences and alcohol tolerance, ethanol and acetaldehyde content was examined in red grapes infested by D. melanogaster and D. suzukii. We assessed mortality and alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activity levels for 2 Drosophila species exposed to ethanol. The study results showed that ethanol content gradually increased as the fruit decayed while being infested by Drosophila. The ethanol content was higher in the presence of D. melanogaster than in the presence of D. suzukii. In the mortality experiment, the LC₅₀ of D. melanogaster adults was approximately 8.0% following exposure to ethanol for more than 6 h, while it was only 2.7% in D. suzukii. Moreover, D. melanogaster adults and larvae all had higher ADH and ALDH activity than D. suzukii exposed to ethanol. Our results suggest that D. melanogaster and D. suzukii may occupy different ecological niches due to their discrepancy in tolerance to environmental ethanol, which is mainly regulated by ADH and ALDH.