Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F ₄₂₀ , and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F ₄₂₀ is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F ₄₂₀ in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F ₄₂₀ , determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F ₄₂₀ .