- Emerging β-lactamase-mediated resistance is threatening the clinical utility of the single most prominent class of antibacterial agents used in medicine, the β-lactams. The diazabicyclooctane avibactam is able to inhibit a wider range of serine β-lactamases than has been previously observed with β-lactamase inhibitors such as the widely prescribed clavulanic acid. However, despite its broad-spectrum activity, variable levels of inhibition have been observed for molecular class D β-lactamases. In order to better understand the molecular basis and spectrum of inhibition by avibactam, we provide structural and mechanistic analysis of the compound in complex with important class A and D serine β-lactamases. Herein, we reveal the 1.7- and 2.0-Å-resolution crystal structures of avibactam covalently bound to class D β-lactamases OXA-10 and OXA-48. Furthermore, a kinetic analysis of key active-site mutants for class A β-lactamase CTX-M-15 allows us to propose a validated mechanism for avibactam-mediated β-lactamase inhibition including a unique role for S130, which acts as a general base. This study provides molecular insights that will aid in the design and development of avibactam-based chemotherapeutic agents effective against emerging drug-resistant microorganisms.