Agrotis segetum Schiff. (Lepidoptera: Noctuidae) is one of the most serious pests of nearly all vegetables in Turkey. In this study, to find a more effective and safe biological control agent, the bacterial flora of Agrotis segetum were investigated in larvae collected from different populations in the vegetable fields of the Black Sea region of Turkey, and 9 bacterial isolates were cultured from these populations. Seven of these bacteria were determined and characterized at the species level and the rest were characterized at the genus level. Morphological, physiological, and biochemical characteristics of the bacterial isolates were determined by conventional and routine techniques. Characterization of the isolates was also performed using the VITEK 32 bacterial identification system. In addition, a 16S rRNA gene sequence was determined for these isolates to query against the NCBI genetic database and to construct a phylogenetic tree using closely related species. Consequently, the isolates were identified as Bacillus cereus (Ags1), Bacillus sp. (Ags2), Bacillus megaterium (Ags3), Enterobacter aerogenes (Ags4), Acinetobacter calcoaceticus (Ags5), Enterobacter sp. (Ags6), Pseudomonas putida (Ags7), Enterococcus gallinarum (Ags8), and Stenotrophomonas maltophilia (Ags9). All isolates were determined the first time from the flora of A. segetum. Furthermore, the insecticidal activities of the bacterial isolates were tested against A. segetum larvae, and Enterococcus gallinarum (Ags8) was found to cause the highest insecticidal activity (60%) 8 days after application.