Vijayabharathi, R., Kumari, B. R., Sathya, A., Srinivas, V., Abhishek, R., Sharma, H. C. and Gopalakrishnan, S. 2014. Biological activity of entomopathogenic actinomycetes against lepidopteran insects (Noctuidae: Lepidoptera). Can. J. Plant Sci. 94: 759-769. The aim of the present study was to identify an efficient broad-spectrum bio-pesticide for the control of lepidopteran insects from microbes in various ecological niches. A total of 111 microbes isolated from various herbal vermi-composts and organically cultivated fields were evaluated for their intracellular metabolites (ICM), extracellular metabolites (ECM) and whole culture (WC) against early instars of lepidopteran insects. Fifteen actinomycete isolates which showed insecticidal activity against 2nd instar Helicoverpa armigera were selected and further screened against Spodoptera litura and Chilo partellus. A significant broad spectrum insecticidal activity was found in the order ECM>ICM>WC against all the insects under laboratory conditions. All these actinomycete isolates also registered significant activity under greenhouse conditions on 2nd instar H. armigera. The actinomycete isolates were identified by 16S rDNA sequencing and matched with Streptomyces species using BLAST search. Among all the 15 isolates, SAI-25 (S. griseoplanus), CAI-155 (S. bacillaris) and BCA-698 (S. albolongus) showed consistent entomopathogenic activity against all the three insects suggesting their potential as broad-spectrum biocontrol agents against other lepidopterans.