Disruption of bacterial cell wall biosynthesis inMycobacterium tuberculosisis a promising target for treating tuberculosis. Thel,d-transpeptidase LdtMt2, which is responsible for the formation of 3 → 3 cross-links in the cell wall peptidoglycan, has been identified as essential forM. tuberculosisvirulence. We optimised a high-throughput assay for LdtMt2, and screened a targeted library of ∼10 000 electrophilic compounds. Potent inhibitor classes were identified, including established (e.g., β-lactams) and unexplored covalently reacting electrophilic groups (e.g., cyanamides). Protein-observed mass spectrometric studies reveal most classes to react covalently and irreversibly with the LdtMt2catalytic cysteine (Cys354). Crystallographic analyses of seven representative inhibitors reveal induced fit involving a loop enclosing the LdtMt2active site. Several of the identified compounds have a bactericidal effect onM. tuberculosiswithin macrophages, one with an MIC50value of ∼1 μM. The results provide leads for the development of new covalently reaction inhibitors of LdtMt2and other nucleophilic cysteine enzymes.