Over 380 host plant species have been known to develop leaf spots as a result of the fungusAlternaria alternata. It is an aspiring pathogen that affects a variety of hosts and causes rots, blights, and leaf spots on different plant sections. In this investigation, the lipopeptides from the B. subtilis strains T3, T4, T5, and T6 were evaluated for their antifungal activities. In the genomic DNA, iturin, surfactin, and fengycin genes were found recovered fromB. subtilisbacterium by PCR amplification. From differentB. subtilisstrains, antifungal Lipopeptides were extracted, identified by HPLC, and quantified with values for T3 (24 g/ml), T4 (32 g/ml), T5 (28 g/ml), and T6 (18 g/ml). To test the antifungal activity, the isolated lipopeptides from the B. subtilis T3, T4, T5, and T6 strains were applied toAlternaria alternataat a concentration of 10 g/ml. Lipopeptides were found to suppressAlternaria alternataat rates of T3 (75.14%), T4 (75.93%), T5 (80.40%), and T6 (85.88%). The T6 strain outperformed the other three by having the highest antifungal activity againstAlternaria alternata(85.88%).Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02922-w.