<jats:title>Abstract</jats:title><jats:p>The structures of the amphiphilic peptide antibiotics herbicolin A and B were determined by application of physical methods, chemical degradation, and partial syntheses. Herbicolin B is a lipodepsinonapeptide with the sequence DH‐Abu‐L‐Thr‐D‐aThr‐D‐Leu‐Gly‐D‐Gln‐Gly‐<jats:italic>N</jats:italic>‐Me‐L‐aThr‐L‐Arg (DH‐Abu = 2,3‐dehydro‐α‐aminobutyric acid). The C‐terminal Arg residue forms a lactone ring with the hydroxy group of L‐Thr, while the N‐terminus is acylated by an (<jats:italic>R</jats:italic>)‐3‐hydroxytetradecanoic acid residue. The main component herbicolin A differs from herbicolin B in an additional D‐glucose moiety linked in an 1‐α‐glycosidic bond to the 3‐hydroxytetradecanoic acid residue. Thus herbicolin A constitutes the first glycolipodepsinonapeptide antibiotic known so far.