Abstract Infections caused by the opportunistic pathogen Mycobacterium abscessus are increasing in prevalence within the cystic fibrosis population. Our limited understanding of this ubiquitous environmental microorganism matched with its intrinsic resistance to most classes of antibiotic, including β -lactams, has left us with an urgent demand for new, more effective therapeutic interventions. De novo antimicrobial drug discovery is a lengthy process and so we have taken the approach of repurposing known antibiotics in order to provide a rapidly implementable solution to a current problem. Here we report a significant step forward in treatment potential for M. abscessus infection by sensitising the organism to the broad spectrum β -lactam antibiotic, amoxicillin, using the non-competitive β -lactamase inhibitor, relebactam. We demonstrate by disk diffusion and broth microdilution assay that this combination works synergistically to inhibit M. abscessus. We also demonstrate the direct non-competitive inhibition of the M. abscessus β -lactamase, Bla Mab using a novel thin-layer chromatography-based assay for β -lactamase inhibition, which is subsequently kinetically validated by spectrophotometric assay using the nitrocefin reporter assay. Finally, we demonstrate the in vitro efficacy of this combination against a collection of M. abscessus clinical isolates, demonstrating the significant therapeutic potential of the amoxicillin and relebactam combination.