To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.European Community’s Seventh Framework ProgramSlovak Research and Development AgencyUK Medical Research CouncilBill and Melinda Gates FoundationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Department of Biology and Biotechnology Lazzaro Spallanzani, University of Pavia, Pavia, ItalyA. N. Bakh Institute of Biochemistry, Russian Academy of Science, Moscow, RussiaInstitut Pasteur, Unité de Microbiologie Structurale, CNRS-UMR3528, Université Paris Diderot, Paris, FranceGlobal Health Institute, Ecole Polytechnique Fédérale de Lausanne, Lausanne, SwitzerlandDepartment of Molecular Medicine, University of Padova, Padua, ItalyCollaborative Drug Discovery, Bayshore Highway, Burlingame, CA, USAFrancis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London, UKFaculdade de Ciências Farmacêuticas (FCFAR), Universidade Estadual Paulista (UNESP), Araraquara, SP, BrasilDepartment of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 6, Mlynská dolina, Bratislava, SlovakiaInstitute of Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 6, Mlynská dolina, Bratislava, Slovak RepublicDepartment of Chemistry, University of Pavia, Pavia, ItalyDepartment of Biology, University of Florence, Sesto Fiorentino, Florence, ItalyInstitut Pasteur de Lille, Center for Infection and Immunity, Lille, FranceFrancis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London, UKGlobal Health Institute, Ecole Polytechnique Fédérale de Lausanne, Lausanne, SwitzerlandDepartment of Biology and Biotechnology Lazzaro Spallanzani, University of Pavia, Pavia, ItalyFaculdade de Ciências Farmacêuticas (FCFAR), Universidade Estadual Paulista (UNESP), Araraquara, SP, BrasilEuropean Community’s Seventh Framework Program: 260872Slovak Research and Development Agency: DO7RP-0015-11UK Medical Research Council: MC_UP_A253_1111Bill and Melinda Gates Foundation: 49852FAPESP: 2011/21232-1CNPq: 140079/2013-0CAPES: 99999.003125/2014-09