<jats:title>ABSTRACT</jats:title> <jats:p> Three families of <jats:named-content content-type="genus-species">Bacillus</jats:named-content> cyclic lipopeptides—surfactins, iturins, and fengycins—have well-recognized potential uses in biotechnology and biopharmaceutical applications. This study outlines the isolation and characterization of locillomycins, a novel family of cyclic lipopeptides produced by <jats:named-content content-type="genus-species">Bacillus subtilis</jats:named-content> 916. Elucidation of the locillomycin structure revealed several molecular features not observed in other <jats:named-content content-type="genus-species">Bacillus</jats:named-content> lipopeptides, including a unique nonapeptide sequence and macrocyclization. Locillomycins are active against bacteria and viruses. Biochemical analysis and gene deletion studies have supported the assignment of a 38-kb gene cluster as the locillomycin biosynthetic gene cluster. Interestingly, this gene cluster encodes 4 proteins (LocA, LocB, LocC, and LocD) that form a hexamodular nonribosomal peptide synthetase to biosynthesize cyclic nonapeptides. Genome analysis and the chemical structures of the end products indicated that the biosynthetic pathway exhibits two distinct features: (i) a nonlinear hexamodular assembly line, with three modules in the middle utilized twice and the first and last two modules used only once and (ii) several domains that are skipped or optionally selected.