Actinomycetes of marine environment are one of the most productive and novel micro organism having potential to produce a large number of substances of economic importance. The current research emphasizes on isolation, screening, physiological and biochemical characterization of actinomycetes form different areas of Bhitarkanika mangrove forest soil and study of their antagonistic effect against few human pathogens. Actinomycetes were isolated by serial dilution followed by plating on actinomycetes isolation agar and starch casein agar media. A total of thirty five (35) actinobacterial isolates were selected on the basis of colony characteristics (colour, morphology) on the above isolation medium and were primarily screened against 10 human pathogens ie., E.coli (ATCC 25922), Pseudomonas aeruginosa (MTCC 741 & ATCC 27583), Klebsiella pneumoniae (MTCC 109 ), Staphylococcus aureus (MTCC 902 & ATCC 25923) Salmonella typhi (MTCC 733), Salmonella enterica (ATCC 35640), Shigella flexneri (MTCC 1457) and Micrococcus luteus (MTCC 1538) by cross streak method. Nineteen isolates showed antagonistic activity against the test pathogens, whose cell free supernatant were again screened by agar well diffusion method. Eight (S1, S7, S8, S16, S21, S22, S24 and S25) isolates were found positive against most tested pathogens were further studied secondarily for their antibacterial activity by agar well diffusion method.