Polyketide synthases type I (PKS-I) gene cluster is responsible for the synthesis of highly assorted group of secondary metabolites such as antimicrobial and anticancer agents. In our study, screening was carried out using degenerate primers to determine the presence of PKS-I gene cluster in endophytic actinomycetes isolated from two medicinal plants Ocmium teniflorum (Tulsi) and Azadirachta indica (Neem). A total of 28 endophytes that were isolated and identified from our previous study were further confirmed through 16S rRNA gene sequencing to exhibit a 99% similarity with Streptomyces sp. The molecular screening using PCR revealed the presence of PKS- I gene with a product size of 750bps in the isolates, FHK-1, FHK-2, FHK-3, FHK-4, FHK-5, FHK-6, FHK-7, FHK-8, FHK-9, FHK-11, FHK-13, FHK-16, FHK-18, FHK-20, FHK-21, FHK-23, FHK,25 and FHK-28. These isolates were further checked for their antimicrobial potential using their crude extracts. They displayed prominent bioactivity against ATCC pathogens, Escherichia coli, Proteus vulgaris, Rhodococcus equi, Staphylococcus epidermidis, Enterococcus faecalis, and Acinetobacter baumanii. Our study revealed that the endophytes from O. tenuiflorum and A. indica are bioactive and versatile harboring the PKS-I gene cluster.