<jats:p><jats:italic>Iso</jats:italic>‐maleimycin, a previously unknown constitutional isomer of the antibiotic maleimycin, has been detected in an extract of <jats:italic>Streptomyces</jats:italic> sp. QL37. Chemical synthesis of both maleimycin (20 % yield over seven steps) and <jats:italic>iso</jats:italic>‐maleimycin, (15 % yield over six steps) allowed access to reference materials for identification. Gas Chromatography coupled Mass Spectrometry (GC‐MS) analysis demonstrated that of the two isomers, only iso‐maleimycin was present in the extract. This finding supports our hypothesis that <jats:italic>iso</jats:italic>‐maleimycin is a biosynthetic intermediate of lugdunomycin. <jats:italic>Iso</jats:italic>‐maleimycin displays low antibiotic activity, with a Minimum Inhibitory Concentration (MIC) value on both <jats:italic>E. coli</jats:italic> and <jats:italic>B. subtilis</jats:italic> of 250 µg/mL.