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The current study aimed to screen bacteriocin producing LAB from different dairy products and evaluation of their biological properties. Initially, 12 (4-chess, 4-curd, and 4-yohurt) LAB species were isolated and only 4 isolates alone were selected based on their clear yellow halo zone around the colonies in the selective medium. The selected 4 isolates were identified based on their morphological and biochemical characteristics. Among them, the strain CH3 have showed better antimicrobial effects on selected human pathogens. The isolated strain CH3 were further identified as Lactococcus lactis strain CH3 (MZ636710) by SEM imaging and 16 s rRNA molecular sequencing. Bacteriocin was extracted from L. lactis strain CH3 and partially purified using 60 % ammonium sulphate and then completely purified by G-50 column chromatography. The purified bacteriocin showed a specific activity of 5859.37 AU/mg in 24.7 % of recovery and 10.9-fold purification. The molecular weight of bacteriocin was 3.5 kDa as observed in SDS-PAGE. The bacteriocin showed sensitivity to proteolytic enzymes and resistance to high temperature, wide range of pH, organic solvents and detergents. FT-IR spectral studies of bacteriocin detected the existence of OH/NH-stretching, CH, and COC and CO bonds. NMR spectrum showed one doublet and 4 various singlet peaks at different ppm, indicating the occurrence of six amino acids in the structure of purified bacteriocin. The purified bacteriocin have shown stronger antimicrobial and anti-biofilm activity against selected human pathogens at 100 μg/mL. SEM showed the evidence of structural deformation and loss of membrane integrity of bacterial cells treated with bacteriocin. Bacteriocin exhibited greater DPPH radical scavenging potential with an EC 50 value of 12.5 μg/mL. Bacteriocin have not shown significant toxicity on normal human dermal fibroblast (NHDF) cells (83.2 % at 100 μg/ mL). Furthermore, in silico studies using molecular modeling and docking were performed to know the proteins involved in antimicrobial action. The results suggests that bacteriocin could be an alternative to combat AMR pathogens and more suitable for food and dairy industries to preserve food without contamination., (Copyright © 2022 Elsevier B.V. All rights reserved.)