Probiotic lactobacilli and its molecules to be used as natural antimicrobials to kill food-borne pathogens as well as therapeutic agents for better development of human gut have drawn amazing attention among consumers. Thus the present study was aimed to screen a probiotic Lactobacillus plantarum (Lp) KJB23 strain for the production of bioactive compound and it was purified using cell adsorption-desorption technique followed by chromatographic techniques. The antilisterial activity of purified compound was not decreased during protease treatment, confirming the non-proteinaceous nature. The compound was identified as 3,4,5,8-tetrahydroxyhexahydro-2H-pyrano[2,3-d][1,2]dioxin-2-one and its molecular weight was found to be 222.1490 Da using HRMS-ESI. This compound has not been reported so far from Lactobacillus sp. The notable bactericidal effect of compound may have potential to be used as antimicrobial agent in food industry as evidenced by pore formation on cell membrane of L. monocytogenes together with its non-cytotoxicity effect. According to our results, the effective non-proteinaceous and non-cytotoxic low molecular weight antibiotic-type compound may have valuable attribute in view of their use in efficient food improvement and can be supportive for developing alternate remedial approaches against food-borne and human pathogens. © 2022 Elsevier B.V., All rights reserved.