Root-knot nematode (RKN), Meloidogyne incognita, inflicts significant greenhouse crop damage, causing over 40% economic losses. While chemical agents effectively control RKN, they leave harmful residue in soil and crops. To address this issue, we investigated soil-derived entomopathogenic fungi (EPF) in this study. We assessed the nematicidal impact of ten EPF species through in vivo and in vitro tests. Beauveria bassiana JEF-503 exhibited nematicidal effectiveness exceeding 65% in both experimental settings. In a semi-field test using greenhouse pots, we experimented with the application of B. bassiana JEF-503 suspension during tomato growth, which is an uncommon practice in comparison to chemical treatments due to residue concerns. B. bassiana JEF-503 was applied during transplantation, and the same application was repeated four weeks later. JEF-503 One-time and abamectin treatments were used as control groups. The JEF-503 two-time application notably reduced root damage compared to single JEF-503 or abamectin treatment. In conclusion, JEF-503 offers a promising, residue-free, and environmentally friendly alternative to chemical agents for RKN management.