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The broad‐spectrum fungal antagonist, Bacillus amyloliquefaciens 6B (BA6B), isolated from the Jakhao coast of Kutch, India, was investigated for its antifungal metabolites using mass spectrometry. The cyclic lipopeptides harvested from the cell‐free fermentation broth of BA6B by acid precipitation and subsequently dissolved in methanol were subjected to liquid chromatography coupled with electrospray ionization mass spectrometry (LC–ESI–MS/MS) for their identification and sequence determination. The 26 types of surfactin variants were identified from the methanolic extract by LC–ESI–MS/MS analysis. Among 26 surfactin species, several new cyclic as well as acyclic surfactin variants based on the variation in the β‐hydroxy fatty acid (β‐OH FA) chain length and/or in amino acid positions 4, 5, 6, and 7 were identified. The mass spectrometric analysis of crude extract also enabled the identification of 11 unique …