Lactobacillus brevis FPTLB3 was isolated from freshwater fish, capable of producing bacteriocin that had broad spectrum of inhibition (3200 AU/ml) against Escherichia coli MTCC 1563, Enterococcus faecalis MTCC 2729, Lactobacillus casei MTCC 1423, Lactobacillus sakei ATCC 15521 and Staphylococcus aureus ATCC 25923. The antimicrobial activity of crude supernatant fluid was stable after heating at 121°C for 60 min and declined thereafter. Stability of antimicrobial activity was observed at pH range of 2.0 to 8.0.