<jats:title>Abstract</jats:title><jats:p>Tuberculosis remains one of the world’s deadliest communicable diseases, novel anti-tuberculosis agents are urgently needed due to severe drug resistance and the co-epidemic of tuberculosis/human immunodeficiency virus. Here, we show the isolation of six anti-mycobacterial ilamycin congeners (<jats:bold>1</jats:bold>–<jats:bold>6</jats:bold>) bearing rare <jats:sc>L</jats:sc>-3-nitro-tyrosine and <jats:sc>L</jats:sc>-2-amino-4-hexenoic acid structural units from the deep sea-derived <jats:italic>Streptomyces atratus</jats:italic> SCSIO ZH16. The biosynthesis of the rare <jats:sc>L</jats:sc>-3-nitrotyrosine and <jats:sc>L</jats:sc>-2-amino-4-hexenoic acid units as well as three pre-tailoring and two post-tailoring steps are probed in the ilamycin biosynthetic machinery through a series of gene inactivation, precursor chemical complementation, isotope-labeled precursor feeding experiments, as well as structural elucidation of three intermediates (<jats:bold>6</jats:bold>–<jats:bold>8</jats:bold>) from the respective mutants. Most impressively, ilamycins E<jats:sub>1</jats:sub>/E<jats:sub>2</jats:sub>, which are produced in high titers by a genetically engineered mutant strain, show very potent anti-tuberculosis activity with an minimum inhibitory concentration value ≈9.8 nM to <jats:italic>Mycobacterium tuberculosis</jats:italic> H37Rv constituting extremely potent and exciting anti-tuberculosis drug leads.