摘要Objective: To isolate strains of Actinomycetes from different locations of Gwalior to evaluate its antimicrobial activity against multidrug resistant pathogenic strains. Method: Soil samples collected from different niche habitats of Gwalior were serially diluted and plated on selective media. Potential colonies were further purified and stored in agar slants and glycerol stocks. Isolates were biochemically characterized and purified isolates were test against pathogenic microorganisms for screening. Isolates with antagonistic properties were inoculated in production media and secondary metabolites or antimicrobial products were extracted. Result: The seven actinomycetes strains showing maximum antibacterial activity were isolated further characterized based on their colony characteristics and biochemical analyses. The isolates were screened for their secondary metabolites activity on three human pathogenic bacteria are Escherichia coli (E. coli), Methicillin-Resistant Staphylococcus aureus (S. aureus) and Vancomycin-Resistant Enterococci (VRE). Discussion: The strain MITS 1005 was found to be more active against the test bacteria.