The entomopathogenic fungus (EPF),Beauveria bassiana, is reported as the most potent biological control agent against a wide range of insect families. This study aimed to isolate and characterize the nativeB. bassianafrom various soil habitats in Bangladesh and to evaluate the bio-efficacy of these isolates against an important vegetable insect pest,Spodoptera litura. Seven isolates from Bangladeshi soils were characterized asB. bassianausing genomic analysis. Among the isolates, TGS2.3 showed the highest mortality rate (82%) against the 2nd instar larvae ofS. lituraat 7 days after treatment (DAT). This isolate was further bioassayed against different stages ofS. lituraand found that TGS2.3 induced 81, 57, 94, 84, 75, 65, and 57% overall mortality at egg, neonatal 1st, 2nd, 3rd, 4th, and 5th instar larvae, respectively, over 7 DAT. Interestingly, treatment withB. bassianaisolate TGS2.3 resulted in pupal and adult deformities as well as decreased adult emergence ofS. litura. Taken together, our results suggest that a native isolate ofB. bassianaTGS2.3 is a potential biocontrol agent against the destructive insect pestS. litura. However, further studies are needed to evaluate the bio-efficacy of this promising native isolatein plantaand field conditions.