Ganfeborole (GSK3036656) inhibits the Mycobacterium tuberculosis leucyl-tRNA-synthetase (mtLeuRS) and is in Phase 2a clinical trials for the treatment of tuberculosis. Here we show that ganfeborole is a time-dependent inhibitor of mtLeuRS (IC50 1 nM) and generates a postantibiotic effect of 77 h at 50xMIC (MIC 0.058 μM) with M. tuberculosis H37Rv, indicating that mtLeuRS is a highly vulnerable drug target and supporting the excellent in vivo efficacy of the drug. Ganfeborole is also a potent time-dependent inhibitor of Escherichia coli LeuRS (ecLeuRS, IC50 2 nM), however no antibacterial activity is observed toward E. coli up to 1 mM ganfeborole despite the observation that less potent ganfeborole analogs have antibacterial activity. To rationalize this observation, we propose that ganfeborole forms a complex with AMP that binds to the ecLeuRS editing site but does not impact aminoacylation. In support, addition of 12.5 μM norvaline generates a ganfeborole MIC of 0.4 μM since ecLeuRS is unable to hydrolyze norvaline-tRNALeu. Additionally, mutations that reduce the affinity and residence time of ganfeborole-AMP on ecLeuRS result in antibacterial activity. We propose that the activity of ganfeborole toward M. tuberculosis is because mtLeuRS is a highly vulnerable target so that only low levels of enzyme need to be inhibited by the ganfeborole-tRNALeu complex in contrast to ecLeuRS, which we previously demonstrated is a low vulnerability target.