The microbial mosquito larvicides Bacillus thuringiensis var. israelensis (Bti) and Lysinibacillus sphaericus (Lsph) are the most widely used biological control agents in mosquito control programs. However, a fast and accurate method to trace the environmental fate of Bacillus biocides released during mosquito control programmes was lacking. We developed a culture-independent technique based on direct DNA extraction from environmental samples such as soil, sediment and water, followed by specific quantifications of Bti and Lsph spores using real-time PCR. These techniques allowed the analysis of the dynamics, persistence, possible accumulation, and dispersal of Bti and Lsph spores applied to control mosquitoes in wetland and urban environments of southern Switzerland.