The endosymbiotic bacteria Wolbachia and the gut microbiome have independently been shown to affect several aspects of insect biology, including reproduction, development, lifespan, stem cell activity and resistance to human pathogens in insect vectors. This research shows that Wolbachia, which reside mainly in the fly germline, affect the microbial species present in the gut of a lab reared strain of Drosophila melanogaster. Fruit flies host two main genera of commensal bacteria – Acetobacter and Lactobacillus. Wolbachia-infected flies have significantly reduced titers of Acetobacter. Analysis of the microbiome of axenic flies fed with equal proportions of both bacteria shows that Wolbachia’s presence is a determinant in the microbiome composition throughout fly development. This effect of Wolbachia on the Drosophila microbiome is host genotype-dependent. To investigate the mechanism of microbiome modulation, the effect of Wolbachia on Imd and ROS pathways, the main regulators of immune response in the fly gut was measured. Wolbachia’s presence did not cause significant gene expression changes of the effector molecules in either pathway. It was also found that Wolbachia slightly reduce the relative length of the acidic region of the gut. However, this observation lacks the robustness necessary to provide a mechanism for the significantly reduced Acetobacter levels. Furthermore, microbiome modulation is not due to direct interaction between Wolbachia and the gut microbes,as confocal microscopy shows that Wolbachia is absent from the gut lumen. These results indicate that the mechanistic basis of the modulation of microbiome composition by Wolbachia is more complex than direct bacteria interaction or Wolbachia’s effect on fly immunity. The findings reported here highlight the importance of considering the gut microbiome composition and host genetic background during Wolbachia-induced phenotypic studies and microbial based-disease vector control strategies.