ABSTRACT Actinomycetes are group of bacteria, which are present widely in ecological diversity. Actinomycetes are the major microbial population in soil which can produce active secondary metabolites. Aim of this study is to isolate varieties of Actinomycetes from different locations to evaluate its antimicrobial activity. Soil samples were collected from different niche habitats of Gwalior (Dry soil from Hostel zone of Madhav Institute of Technology and Science (MITS), Cancer hospital territory, IIITM ground, forest areas of Sanjeevani nursery) and used for actinomycetes isolation. The seven actinomycetes isolates were isolated from different soil samples based on their colony morphology, mycelium colouration and diffusion of pigmentation. The isolates were analysed for their biochemical activity and found that all the isolates were positively active for catalase, starch and casein utilization. The isolates were screened for their secondary metabolites activity on three human pathogenic bacteria are Escherichia coli, Staphylococcus epidermis and Vencomycin resistant Enterococci (vre). The isolate 5 was found to be more active against the test bacteria. The isolate 5 was further tested for its zone of inhibition and found maximum activity (14 mm) against the test bacteria. It was concluded that actinomycetes isolate 5 had potentials as sources of new antibacterial compounds against pathogenic microorganisms (unicellular Gram positive and Gram negative bacteria and drug resistance microorganism) to humans.