The burden of increased antimicrobial resistance in microorganisms has complicated disease management and healthcare systems. Mechanisms like enzymatic modification of antibiotics, biofilm formation and genetic mutations enable pathogens to evade antibiotic treatments. In this view, alternatives for antimicrobial agents from natural sources are a subject of concern and should be explored immediately. Bacteriocins are antimicrobial peptides with targeted activity against specific bacteria. They are effective at low concentrations (Soltani et al., 2022) and demonstrate both narrow- and broad-spectrum activity. Bacteriocins selectively target and inhibit pathogenic bacteria, minimizing the development of resistance and preserving beneficial microbiota through quorum sensing. Their molecular weight ranges from <5 to 80 kDa with a smaller number of amino acids according to RiPP nomenclature of bacteriocin classification. Our study has emphasized isolation of novel peptides from the species of Levilactobacillus brevis through microbiological techniques and their purification using Prep-HPLC and characterization by biomolecular techniques—High-Performance Thin-Layer Chromatography (HPTLC), Proton-Nuclear Magnetic Resonance (H-NMR) and MALDI-TOF. The antimicrobial activity of purified bacteriocin was determined using an agar-well assay against pathogenic organisms—Streptococcus mutans, Escherichia coli and Candida auris. The isolated peptide is characterized as a Class II bacteriocin with amino acid no. 8 (partial N-terminal sequence) and size 0.86 kDa (in accordance with RiPP nomenclature of bacteriocin classification). Peptides displayed potential antimicrobial effects with zones of inhibition against Escherichia coli at 0.2 cm, Streptococcus mutans at 0.3 cm and Candida albicans at 0.12 cm; p value = 0.00004 (p≤0.05). This is statistically significant. There is a current need to explore other such peptides and further apply them in pharmacological, food and biomedical sectors to strengthen their effectiveness against antimicrobial resistance. The current research data adds to the need to search for such alternate molecules.