The microbial communities that live inside of them have an impact on many elements of animal ecology and physiology, which in turn has an impact on the health, survival, and reproductive fitness of the organisms. Gut bacterial diversity within an organism is influenced by both biotic and abiotic causes. In nature, seasonal and altitudinal change are significant forces that also influence the variety of the bacteria in the gut. Determining the effects of altitude and seasonal fluctuation on gut microbial diversity in three sympatric species of Drosophila, including D. malerkotliana, D. bipectinata, and D. nasuta in Chamundi Hill, is the goal of the current study. Here, using 16S rRNA gene sequencing, we examined the quantitative changes in intestinal microbiota in three sympatric species of Drosophila collected at various altitudes and seasons. Two species from the Acetobacter species group, Acetobacter pomorum and Acetobacter tropicalis, three species, Lactobacillus brevis, Lactobacillus fructivorans, and Lactobacillus plantarum, found to belong to the Lactobacillus species were found in all three altitudes and seasons examined in the current study. The relative abundance of each of the aforementioned microbial species fluctuates significantly depending on height and season, according to further investigation. Further relative abundance of gut bacteria also varied between sympatric Drosophila species in different altitudes and seasons. Except A. pomorum all other gut bacteria were found to be significantly greater at low altitude compared to middle and high altitudes. Among the seasons, the density of L. fructivorans, A. tropicalis and A. pomorum were found to be greatest in monsoon season followed by premonsoon and post monsoon seasons. Thus theseresults imply that there is a significant effect of altitude and season on the abundance of gut bacteria in three sympatric Drosophila species of Chamundi hills.