A total of 61 Bacillus thuringiensis strains were collected from 264 soil samples representing different forest ecosystem in Southern zone of Andhra Pradesh in Chittoor, Kadapa and Nellore districts during 2014-2016. These strains were tested for their efficacy against third instar larvae of Spodoptera litura in laboratory bioassay studies. A series of 0.00 to 53.33; 0.00 to 20.00; 0.00 to 30.00; 0.00 to 30.00 and 0.00 to 23.33 per cent larval mortality was observed at 72, 96, 120, 144 and 168 hours after treatment, respectively. A cumulative mortality of 0 to 100 per cent was observed among the 61 strains collected from forest ecosystem. The Bt strain F493 recorded highest mortality of 100 per cent, followed by F468 (86.67%) which were comparable with standard strain HD-1 for their efficacy. The other strains F287 (76.67%), F504 (76.67%) were proved next best to F493, HD-1 and F468 for their efficacy which were statistically on par with each other.