AbstractThe caseinolytic protease complex ClpP1P2 is crucial for protein homeostasis in mycobacteria and stress response and virulence of the pathogens. Its role as a potential drug target for combating tuberculosis (TB) has just begun to be substantiated in drug discovery research. We conducted a biochemical screening targeting the ClpP1P2 using a library of compounds phenotypically active against Mycobacterium tuberculosis (Mtb). The screening identified a phenyl ester compound GDI‐5755, inhibiting the growth of Mtb and M. bovis BCG, the model organism of mycobacteria. GDI‐5755 covalently modified the active‐site serine residue of ClpP1, rendering the peptidase inactive, which was delineated through protein mass spectrometry and kinetic analyses. GDI‐5755 exerted antibacterial activity by inhibiting ClpP1P2 in the bacteria, which could be demonstrated through a minimum inhibitory concentration (MIC) shift assay with a clpP1 CRISPRi knockdown (clpP1‐KD) mutant GH189. The knockdown also remarkably heightened the mutant's sensitivity to ethionamide and meropenem, but not to many other TB drugs. On the other hand, a comparative proteomic analysis of wild‐type cells exposed to GDI‐5755 revealed the dysregulated proteome, specifically showing changes in the expression levels of multiple TB drug targets, including EthA, LdtMt2, and PanD. Subsequent evaluation confirmed the synergistic activity of GDI‐5755 when combined with the TB drugs to inhibit mycobacterial growth. Our findings indicate that small‐molecule inhibitors targeting ClpP1P2, when used alongside existing TB medications, could represent novel therapeutic strategies.