The composition of microbiota affects different traits of Drosophila throughout its life cycle and represents an important part of the adaptive response to environmental changes, such as increased levels of accumulated heavy metals in their habitats. We investigated the effects of lead added to the feeding substrate on the microbiota diversity in the fruit flies, Drosophila melanogaster and Drosophila subobscura. We compared the bacterial diversity of wild-caught flies and their progeny reared under standard laboratory conditions. We analyzed the shifts in bacterial diversity in the experimental groups of flies reared for 13 generations on standard and lead(II) acetate (Pb(CH3COO)2), Pb acetate-saturated substrates. Identification of the main bacterial genera was performed by next-generation sequencing (NGS) of the V3-V4 variable regions of the 16S rRNA gene. Results indicate significant species-specific differences in bacterial composition between natural and laboratory populations, and between the substrates. Diversity increased in both species under prolonged exposure to lead-polluted substrate, suggesting a potential adaptive response to an environmental stress.