Actinobacteria is a goldmine for the discovery of abundant secondary metabolites with diverse biological activities. This study explores antimicrobial biosynthetic potential and diversity of actinobacteria from Pobitora Wildlife Sanctuary and Kaziranga National Park of Assam, India, lying in the Indo-Burma mega-biodiversity hotspot. A total of 107 actinobacteria were isolated, of which 77 exhibited significant antagonistic activity. 24 isolates tested positive for at least one of the polyketide synthase type I, polyketide synthase type II or non-ribosomal peptide synthase genes within their genome. Their secondary metabolite pathway products were predicted to be involved in the production of ansamycin, benzoisochromanequinone, streptogramin using DoBISCUIT database. Molecular identification indicated that these actinobacteria predominantly belonged to genus Streptomyces, followed by Nocardia and Kribbella. 4 strains, viz. Streptomyces sp. PB-79 (GenBank accession no. KU901725; 1313 bp), Streptomyces sp. Kz-28 (GenBank accession no. KY000534; 1378 bp), Streptomyces sp. Kz-32 (GenBank accession no. KY000536; 1377 bp) and Streptomyces sp. Kz-67 (GenBank accession no. KY000540; 1383 bp) showed ~89.5% similarity to the nearest type strain in EzTaxon database and may be considered novel. Streptomyces sp. Kz-24 (GenBank accession no. KY000533; 1367 bp) showed only 96.2% sequence similarity to S. malaysiensis and exhibited minimum inhibitory concentration of 0.024 µg/mL against methicilin resistant Staphylococcus aureus ATCC 43300 and Candida albicans MTCC 227. This study establishes that actinobacteria isolated from the poorly explored Indo-Burma mega-biodiversity hotspot may be an extremely rich reservoir for production of biologically active compounds for human welfare.