In the present study, three entomopathogenic strains of Bacillus sphaericus (MBI5, MBI6, and MBI7) were isolated from greenhouse pest and larval habitat of Istanbul, Turkey. All of the MBI groups tested in laboratory bioassays were able to kill larvae, but not pupae and adults of Culex spp. in water. The most efficient larvicidal activity of the strains was found in the presence of two toxin genes, binA and binB for MBI5, MBI6, and MBI7 strains. Each B. sphaericus strains identified in this study were characterized as unique and novel in terms of fatty acid methyl ester (FAME) profiles and 16S rRNA sequencing data. The results of this study suggest that the three strains of B. sphaericus may be new sources of potential biocontrol agent of mosquito.