Triphenylmethane dyes are carcinogenic and widely used in the aquaculture and textile industries. Bioremediation, the use of microorganisms to degrade xenobiotic compounds, may be a more efficient alternative to conventional treatment methods for dye contaminated waste. To identify genes involved in triphenylmethane dye decolorization by mycobacteria, a transposon mutant library of Mycobacterium smegmatis mc² 155 was created and screened for mutants unable to decolorize the triphenylmethane dye Malachite Green. One gene identified was fbiC, which is essential for the biosynthesis of the electron carrier, coenzyme F₄₂₀. Also identified was MSMEG_2392, belonging to a superfamily without annotated function. High Pressure Liquid Chromatography revealed that F₄₂₀ was absent in both mutant strains, indicating that, like fbiC, MSMEG_2392 is required for the biosynthesis of coenzyme F₄₂₀ and this cofactor is likely the electron donor for the reduction of Malachite Green. This is the first report of this coenzyme in dye decolorization.