INTRODUCTION: Surfactins, a class of lipopeptide biosurfactants secreted by plant growth-promoting rhizobacteria (PGPR), have garnered significant attention due to their dual functionality in promoting plant growth and controlling plant diseases. Their potential as biopesticides is underscored by their unique physicochemical properties and biological activities. However, the practical application of surfactin is currently limited by its low yield in natural strains. METHODS: This study aimed to optimize the culture conditions for RESULTS: The study revealed that nitrogen sources and amino acid supplementation were pivotal factors influencing surfactin yield. Compared to the baseline, these factors resulted in a remarkable 5.94-fold increase in surfactin production. Furthermore, a positive correlation was established between surfactin yield and biocontrol efficacy. Enhanced surfactin yield was associated with improved antifungal activity, biofilm formation, and rhizosphere colonization capacity of DISCUSSION: These findings provide novel insights into the practical application of surfactin and establish a scientific foundation for the development of innovative and eco-friendly antifungal agents suitable for agricultural use. The results demonstrate that optimizing culture conditions can significantly enhance surfactin yield and biocontrol efficacy, thereby highlighting the potential for sustainable agricultural practices.