Gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae), is one of the most serious pests affecting fruit, forest, industrial, and foliage plants worldwide. The aim of this study was to isolate and characterize pathogenic bacteria from this pest and determine their pathogenicity. L. dispar larvae were collected from various populations in the forest areas of the Black Sea region of Turkey in May and June of 2008 and 2009, and 9 culturable bacterial isolates were obtained from these larvae. Morphological and physiological characteristics of bacterial isolates were determined by conventional and routine techniques, and biochemical properties and metabolic enzyme profiles were revealed using API20E and Phoenix 1000A panel test systems. Additionally, a 16S rRNA gene sequence analysis was performed to identify the isolates at the molecular level and to query against the NCBI genetic database. Consequently, 7 of the bacterial isolates were determined and characterized at the species level while the rest were characterized at the genus level. From the results of all tests, the isolates were identified as Pantotea agglomerans (Lyd1), Klebsiella sp. (Lyd2), Acinetobacter calcoaceticus (Lyd3), Serratia marcescens (Lyd4), Erwinia sp. (Lyd5), Bacillus thuringiensis (Lyd6), B. thuringiensis (Lyd7), B. thuringiensis (Lyd8), and B. thuringiensis (Lyd9). Of all bacterial isolates tested for insecticidal activity, 2 of the B. thuringiensis isolates (Lyd6 and Lyd9) were found to cause the highest insecticidal activity (90%) within 10 days of application. Furthermore, 100% insecticidal activity was observed with a crystal-spore mixture of the same isolates against Lymantria dispar larvae within 10 days.