Two beta-lactams, cefoxitin and imipenem, are part of the reference treatment for pulmonary infections with Mycobacterium abscessus. M. abscessus has recently been shown to produce a broad-spectrum beta-lactamase, Bla Mab , indicating that the combination of beta-lactams with a Bla Mab inhibitor may improve treatment efficacy. The objectives of this study were to evaluate the impact of Bla Mab production on the efficacy of beta-lactams in vitro and to assess the benefit of Bla Mab inhibition on the activity of beta-lactams intracellularly and in an animal model. Methods: We analysed the mechanism and kinetics of Bla Mab inactivation by avibactam, a non-beta-lactam beta-lactamase inhibitor currently in Phase III of development, in combination with ceftazidime for the treatment of serious infections due to Gram-negative bacteria. We then deleted the gene encoding Bla Mab to assess the extent of Bla Mab inhibition by avibactam based on a comparison of the impact of chemical and genetic inactivation. Finally, the efficacy of amoxicillin in combination with avibactam was evaluated in cultured human macrophages and in a zebrafish model of M. abscessus infection. Results: We showed that avibactam efficiently inactivated Bla Mab via the reversible formation of a covalent adduct. An inhibition of Bla Mab by avibactam was observed in both infected macrophages and zebrafish. Conclusions: Our data identify avibactam as the first efficient inhibitor of Bla Mab and strongly suggest that beta-lactamase inhibition should be evaluated to provide improved therapeutic options for M. abscessus infections.