Tuberculosis remains a leading cause of death from a single bacterial infection worldwide. Efforts to develop new treatment options call for expansion into an unexplored target space to expand the drug pipeline and bypass resistance to current antibiotics. Lipoamide dehydrogenase is a metabolic and antioxidant enzyme critical for mycobacterial growth and survival in mice. Sulfonamide analogs were previously identified as potent and selective inhibitors of mycobacterial lipoamide dehydrogenase in vitro but lacked activity against whole mycobacteria. Here we present the development of analogs with improved permeability, potency, and selectivity, which inhibit the growth of Mycobacterium tuberculosis in axenic culture on carbohydrates and within mouse primary macrophages. They increase intrabacterial pyruvate levels, supporting their on-target activity within mycobacteria. Distinct modalities of binding between the mycobacterial and human enzymes contribute to improved potency and hence selectivity through induced-fit tight binding interactions within the mycobacterial but not human enzyme, as indicated by kinetic analysis and crystallography.