ABSTRACT The use of lactic acid bacteria (LAB) as biopreservatives to control fungal spoilage in food is a promising strategy in terms of natural food preservation. In this study, 300 LAB were isolated from different sources and screened for antifungal activity in vitro against 9 spoilage fungi. Moreover, the most active strain Lactiplantibacillus pentosus 22B was utilized in yogurt as a biopreservative against fungal spoilage. Characterization of the antifungal compounds based on fungal response to concentrated extract after enzymatic treatments, GC-MS, and headspace GC-MS showed that L. pentosus 22B potentially produced a variety of active compounds including organic acids, peptidic compounds, fatty acids, volatile compounds, and hydrogen peroxide. The application of cell free-supernatant produced by L. pentosus 22B in yogurt showed its ability to prevent fungal growth during 22 days at 25°C. This LAB strain is a potential candidate for yogurt biopreservation based on the antifungal activity of L. pentosus in yogurt which has been experimentally confirmed for the first time.