The essential mycobacterial enzyme phosphopantetheinyl transferase (PptT) is a potential target for antituberculosis drugs. We here report the identification of (pyridin-3-ylmethyl)-substituted thioquinazolinones as hits in a screen against PptT and a structure-activity relationship (SAR) study of the chemotype. The resulting thioquinazolinones exert on-target whole-cell activity against Mtb in axenic culture and in macrophages with efficacy comparable to that of clinically used antituberculosis drugs. Increased susceptibility of a pptT hypomorphic strain of Mtb, resistance of PptT Trp170 mutant Mtb strains, and analysis of relevant mutations corroborated that the thioquinazolinones are on target, do not inhibit growth of other bacteria, are not cytotoxic to mammalian cells, did not bind to most mammalian receptors and ion channels tested, and did not react with glutathione. Mtb rapidly took up the thioquinazolinones and methylated them, affording products inactive against recombinant PptT.