SummaryAspergillus flavus and Penicillium spp. are the most common spoilage fungi in the ready‐to‐eat, intermediate‐moisture Thai curry of Kaeng‐Tai‐Pla‐Haeng, which causes serious economic loss and public health concerns. The use of lactic acid bacteria (LAB) with antifungal activity has been a growing interest in food bio‐preservation. In this study, the antifungal efficacy of Lactiplantibacillus plantarum 124 (L. plantarum 124) against the spoilage fungi Aspergillus flavus and Penicillium sp. was evaluated. The cell‐free supernatants (CFSs) of L. plantarum 124 showed strong inhibition against A. flavus and Penicillium sp., with MIC ranging from 11.25 to 22.5 mg/mL. The significant loss of the antifungal activity was observed by pH neutralization, whereas either heat or proteinase K treatment showed no influence. The major compounds present in CFSs were lactic acid (49.57%) and acetic acid (25.85%). Others, such as 2,3‐dimethyl‐5‐ethylpyrazine (6.07%), 2,5‐dimethyl‐3‐propylpyrazine (2.10%), Cyclo(D‐Leu‐L‐Pro) (2.30%), Ethyl 5‐oxopyrrolidine‐3‐carboxylate (1.84%), and palmitic acid (1.80%) were detected at low relative amount. As observed by electron microscopy, the CFSs secreted by L. plantarum 124 caused severe damage to cell membranes, and organelles of A. flavus and Penicillium sp. In addition, L. plantarum 124 supernatant demonstrated no cytotoxicity on Vero cells and it could extend the food product's shelf‐life from 7 days to 21 days without any commercial preservatives. These results demonstrate that antifungal metabolites secreted by L. plantarum 124 are candidates for the development of bio‐preservative agents.