Cysteine is an important amino acid in the redox defense of Mycobacterium tuberculosis, primarily as a building block of mycothiol. Genetic studies have implicated de novo cysteine biosynthesis in pathogen survival in infected macrophages, in particular for persistent M. tuberculosis. Here, we report on the identification and characterization of potent inhibitors of CysM, a critical enzyme in cysteine biosynthesis during dormancy. A screening campaign of 17 312 compounds identified ligands that bind to the active site with micromolar affinity. These were characterized in terms of their inhibitory potencies and structure-activity relationships through hit expansion guided by three-dimensional structures of enzyme-inhibitor complexes. The top compound binds to CysM with 300 nM affinity and displays selectivity over the mycobacterial homologues CysK1 and CysK2. Notably, two inhibitors show significant potency in a nutrient-starvation model of dormancy of Mycobacterium tuberculosis, with little or no cytotoxicity toward mammalian cells.