On using the streptomycin-starved 18b strain as a model for nonreplicating Mycobacterium tuberculosis, we identified a 5-nitrothiophene compound as highly active but not cytotoxic. Mutants resistant to 5-nitrothiophenes were found be cross-resistant to the nitroimidazole PA-824 and unable to produce the F420 cofactor. Furthermore, 5-nitrothiophenes were shown to be activated by the F420-dependent nitroreductase Ddn and to release nitric oxide, a mechanism of action identical to that described for nitroimidazoles.