Screening of a diversity-oriented compound library led to the identification of two 6,11-dioxobenzo[f]pyrido[1,2-a]indoles (DBPI) that displayed low micromolar bactericidal activity against the Erdman strain of Mycobacterium tuberculosis in vitro. The activity of these hit compounds was limited to tubercle bacilli, including the nonreplicating form, and to Mycobacterium marinum. On hit expansion and investigation of the structure activity relationship, selected modifications to the dioxo moiety of the DBPI scaffold were either neutral or led to reduction or abolition of antimycobacterial activity. To find the target, DBPI-resistant mutants of M. tuberculosis Erdman were raised and characterized first microbiologically and then by whole genome sequencing. Four different mutations, all affecting highly conserved residues, were uncovered in the essential gene rv0338c (ispQ) that encodes a membrane-bound protein, named IspQ, with 2Fe-2S and 4Fe-4S centers and putative iron-sulfur-binding reductase activity. With the help of a structural model, two of the mutations were localized close to the 2Fe-2S domain in IspQand another in transmembrane segment 3. The mutant genes were recessive to the wild type in complementation experiments and further confirmation of the hit-target relationship was obtained using a conditional knockdown mutant of rv0338c in M. tuberculosis H37Rv. More mechanistic insight was obtained from transcriptome analysis, following exposure of M. tuberculosis to two different DBPI; this revealed strong upregulation of the redox-sensitive SigK regulon and genes induced by oxidative and thiol-stress. The findings of this investigation pharmacologically validate a novel target in tubercle bacilli and open a new vista for tuberculosis drug discovery.