Peptidoglycan synthesis is an underutilized drug target in (). Diazabicyclooctanes (DBOs) are a class of broad-spectrum β-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the β-lactamase, and multiple peptidoglycan transpeptidases (PonA1, Ldt, Ldt, Ldt, and Ldt). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except Ldt. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC ( 9.2 ± 0.9 μM, / 5600 ± 560 M s) and similar to clavulanate ( 3.3 ± 0.6 μM, / 8400 ± 840 M s); however, durlobactam had a lower turnover number ( = /) than clavulanate (1 and 8, respectively). values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and Ldt. Antibiotic susceptibility testing was conducted on 11 isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 μg/mL, similar to the ranges for meropenem (1-32 μg/mL) and imipenem (0.5-64 μg/mL). In β-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel β-lactamase inhibitors that target BlaC and peptidoglycan transpeptidases.