With the enhanced use of chemical preservatives and the survival of foodborne pathogens, lactic acid bacteria and its metabolic product have received increased attention as a potential food preservative. Even though the antibacterial property of lactic acid bacteria is well known, its antifungal property and application in food preservation have not been widely exploited. Considering these facts, this study was conducted to investigate the antifungal properties of three human gut lactic acid bacterial isolates, Lactobacillus plantarum AL1, Lactobacillus fermentum AL2, and Weissella confusa AL3 antifungal activity of the isolates were studied against the aflatoxigenic Aspergillus flavus MTCC 2798, using the agar overlay method and inhibition of fungal mycelium (co-culture method). These LAB isolates were also potent producers of antimicrobial compounds like lactic acid, hydrogen peroxide, and diacetyl. GC–MS analysis of the methanolic extract of the three LAB isolates detected two antifungal bioactive compounds like cyclo (leucyloprolyl) and 1,2-benzenedicarboxylic acid in extracts. The hemolysis results provided an important insight that the human gut lactic acid bacteria can be used as protective cultures in the biopreservation. The antifungal activity of human gut LAB AL1, AL2, and AL3 against aflatoxigenic Aspergillus flavus MTCC 2798 and its ability extend the shelf life of peanuts prove as a natural preservative for food products.