Bacillus thuringiensis subsp. israelensis (Bti) is the most widely used biopesticide against mosquitoes and blackflies, with a history of high specificity and efficacy. However, interactions of Bti with native microfauna in the environment are poorly understood. High and low Bti (VectoBac G) applications, in addition to an untreated control, were assigned to replicate ?1 m^2? mesocosms in a completely randomized design under field conditions. The abundance of Culex spp., phytoplankton, sestonic particulates, nutrients and other water quality parameters were assessed throughout this large-scale 82-day field study. Bacterial communities present in the water column were assessed by next generation sequencing of 16S rRNA genes, comparing treatments and sampling dates. The high Bti application rate significantly reduced mosquito abundance, phytoplankton biomass, sestonic particulates, nutrients, and other physicochemical variables in the water, and was also associated with higher bacterial diversity. Beta diversity analysis revealed that bacterial communities in the water column were influenced significantly by the high Bti treatment. Together, our results demonstrate significant dose-dependent changes to aquatic ecosystem chemistry and bacterial community composition associated with Bti application.