Lactic acid bacteria (LAB) play an important role as natural food preservatives. However, the characterization of the variety of their metabolites is limited. The increasing societal demand for less processed food products, while conserving those products’ quality, safety and shelf-life, has raised the question of chemical preservative replacement. In this context, the LAB, as well as their metabolites, are alternatives of interest for use in food as bioprotective tools to fight microbial pathogen growth answering to consumer demands. The aim of this study was to determine the production of specific metabolites of L.rhamnosus, L.brevis, L.plantarum and L.gasseri by an optimized liquid chromatography with an ultraviolet/diode detection method and to investigate their potential antimicrobial activity against specific food pathogens. Based on the results of this study, the main metabolites detected in L.brevis were DL-p-Hydroxyphenyllactic acid (OH-PLA) (103.4 ppm) and vanillic acid (2.59 ppm), while OH-PLA (216.2 ppm), salicylic acid (19.0 ppm), vanillic acid (3.7 ppm), ferulic acid (6.9 ppm), benzoic acid (4.2 ppm) and 4-Hydrocinnamic acid (1.4 ppm) were identified in the L.plantarum strain, OH-PLA (147.6 ppm) and ferulic acid (4.9 ppm) were detected in L.rhamnosus and L.gasseri includes DL-p-Hydroxyphenyllactic acid (OH-PLA) (253.0 ppm), 1,2-dihydroxybenzene (12.68 ppm) and benzoic acid (7.83 ppm). Also, investigated the antimicrobial activity of the obtained cell-free-supernatants-CFSs incubation of the four LAB strains against pathogenic microbial species (Salmonella enterica ATCC14028; Staphylococcus aureus ATCC29213; Escherichia coli ATCC25922; Klebsiella pneumoniae ATCC700603). All obtained CFSs from LAB cultures showed significant inhibitory effects on the tested Gram negative and positive pathogens. The isolated LAB inhibited the growth of the tested pathogenic strains successfully, indicating that the addition of LAB strains in commercial food products may provide effective protection against infections caused by specific pathogens. This study provides alternative approaches for the molecules involved in the antimicrobial activity of food microorganism fermentation.