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Fish mucus has been recognized as the potential source of antimicrobial proteins and peptides, which is of interest to provide the front line defence against numerous invading pathogens. The antimicrobial peptides (AMPs) have been reported to possess important ecological and biological functions. Fishes are always in intimate contact with the environment containing diverse and higher concentrations of viruses and bacteria. This study reports the isolation and characterization of a potent antimicrobial peptide, from the skin mucus of bacterial infected fishes collected from different fresh water bodies of Kashmir valley. The fish mucus was subjected to column chromatographic techniques for isolation and purification using gel filtration chromatography. The peptide was demonstrated to be single protein band by SDS-PAGE, with the apparent molecular weight of 20 kDa. The peptide exhibited the antimicrobial property as revealed by disc diffusion technique against three pathogenic strains like Klebsiella pneumoniae, Staphylococcus aureus and E.coli. The purified AMP presented the potential anti-microbial activity with all the three strains of bacteria and the maximum inhibition was recorded against Klebsiella pneumonia (19mm). The broad antimicrobial activities of isolated antimicrobial peptide suggest that it contributes to the innate host defence system in bacterial infected fish.