Bacteriocins are proteins secreted by many species of bacteria to inhibit other bacteria, thus eliminating competitors to gain resources. Bacteria from the Lactobacillus group are known for their applications as probiotics and food preservatives. They have earned a reputation for producing substances that inhibit the growth of other microorganisms, which include organic acids, diacetyl, and bacteriocins. Produced by the ribosomes, bacteriocins are cationic proteins that inhibit other bacteria coexisting within a shared ecological habitat. Due to their potential uses in a variety of applications large-scale production of Bacteriocins would be necessary. The study aimed to identify and characterize Lactobacillus bacteria that produce potent bacteriocins and to analyze the antimicrobial activity and stability of the isolated bacteriocin under various physical and biochemical conditions. A total of 50 samples including buffalo dung, cheese, and rhizospheric region of plants were screened to isolate 8 Lactobacillus Li-1, Li-2, Li-3, Li-4, Li-5, Li-6, Li-7, and Li-8, confirmed by gram staining and other biochemical tests. The cell free supernatant from the Li-3 strain showed higher inhibition of Escherichia coli and Staphylococcus aureus, as compared to the other isolated strains. Li-3 strain was further identified as Levilactobacillus brevis strain ABRIINW-K by 16S rRNA gene sequencing. The bacteriocin isolated from this strain is a thermostable peptide (~6kDa), which is characteristic of class II bacteriocins, with potent antibacterial activity against Lactobacillus rhamnosus, Escherichia coli, and Salmonella enterica.