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Bacteriocins from lactic acid bacteria have attracted considerable attention as natural alternatives to conventional antimicrobial agents. Weissellicin LM85, a bacteriocin purified from Weissella confusa LM85, has been less extensively studied in terms of its mechanism of action and potential applications. In this study, purified weissellicin LM85 exhibited potent inhibitory effects against Gram-positive bacteria, with minimum inhibitory and bactericidal concentrations determined against Micrococcus luteus MTCC106. Time-kill assays and fluorescence staining indicated a concentration-dependent reduction in cell viability, accompanied by membrane disruption. Further analyses revealed potassium ion efflux, dissipation of membrane potential (Δψ) and pH gradient (ΔpH), genomic DNA fragmentation, and pronounced morphological alterations in target cells. These findings are strongly suggestive of membrane-targeted bactericidal activity, likely involving pore-forming effects. In addition, weissellicin LM85 inhibited both growth and biofilm formation of Salmonella enterica subsp. enterica serovar Typhimurium ATCC13311 and Staphylococcus aureus subsp. aureus ATCC25923. Mechanistic analyses revealed the disruption of cell membrane integrity, leakage of potassium ions, cytoplasmic contents, and non-specific DNA degradation, indicating a multifaceted antibacterial mode of action. These findings highlight weissellicin LM85 as a promising natural antimicrobial with potential applications in food preservation and the control of foodborne pathogens and biofilm-associated infections. Further studies on cytotoxicity and in vivo efficacy are required to advance its practical application. Full article