Alternaria alternata is a fungus that has been recorded to cause leaf spots on over 380 host species of plant. It is an opportunistic pathogen on numerous hosts causing leaf spots, rots, and blights on many plant parts. This study aimed to investigate the antifungal activity of the lipopeptides from strains (T3, T4, T5, and T6) of B. subtilis were studied. The genomic DNA was isolated from B. subtilis bacteria and confirmed the presence of iturin, surfactin, and fengycin genes by PCR amplification. Antifungal Lipopeptides were extracted, identified by HPLC, and quantified from B. subtilis strains with the values for T3 (24µg/ml), T4 (32µg/ml), T5 (28µg/ml) and T6 (18µg/ml). The extracted lipopeptides from T3, T4, T5, and T6 strains of B. subtilis at 10µg/ml of media were used against Alternaria alternata to check the antifungal activity. The rate of inhibition using lipopeptides against Alternaria alternata was recorded as T3 (75.14%), T4 (75.93%), T5 (80.40%), and T6 (85.88%). Among the four strains, the T6 strain (85.88%) showed the highest antifungal activity against the Alternaria alternata.