A bacteriocin-producing strain Streptococcus bovis J2 40-2 was isolated from traditional fermented milk ‘Dahi’ in Bangladesh. Despite its narrow antimicrobial spectrum, it showed strong antimicrobial activity against extremely challenging and problematic organisms in foods, such as Listeria monocytogenes. Bacteriocin was sensitive to several proteolytic enzymes and showed antimicrobial activity over a wide pH range of 2.0–10.0. It was stable when heated to 110°C for 20 min, but lost 25% of its activity when heated to 121°C for 15 or 20 min. Optimum bacteriocin production (5600 AU/mL) was achieved when the strain was cultured at 37°C for 24 h in MRS medium rather than in TYLG, GM17, or skim milk medium. Bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation), dialysis (cut-off MW: 3500) and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that bacteriocin had a molecular weight of approximately 4.5 kDa.