Aquaculture is one of the major food-producing sectors in the world. Disease control and management in aquaculture are one of the major constraints in aquaculture development as fish pathogens are becoming resistant to conventional therapeutics. Actinomycetes are one of the most economical and biotechnologically important microbes and have proven themselves as reliable sources of novel bioactive compounds. The objective of the present study is to isolate, screen and to characterize the actinomycetes isolates for antibacterial activity against the fish and shellfish pathogens. In this study, terrestrial soil samples were collected from different places (unexplored/ under-explored regions) of Vellore, Tamil Nadu, India, serially diluted and inoculated onto Actinomycetes Isolation agar (AIA) and Starch Casein agar (SCA). The pure colonies were further sub-cultured and screened for their antibacterial activity by well diffusion method against selected fish bacterial pathogens Edwardsiella tarda, Aeromonas caviae, Vibrio harveyi and Vibrio anguillarum.The isolate EDNK3 showed a wide range of activity against all the pathogens tested. The morphological, physiological and biochemical characterization of the potential isolate was carried out based on which the isolate EDNK3 is assumed to belong to the genus Streptomyces. Based on the results it can be concluded that the isolate EDNK3 from the unexplored regions of Vellore soil has a broad spectrum antibacterial activity against the fish and shellfish pathogens. © RJPT All right reserved.