The combination of the monobactam, aztreonam, and the non-β-lactam β-lactamase inhibitor, avibactam, is currently in clinical development for the treatment of serious infections caused by metallo-β-lactamase (MBL)-producing Enterobacteriaceae, a difficult to treat subtype of carbapenem-resistant Enterobacteriaceae for which therapeutic options are currently very limited. The present study tested clinically significant isolates of Enterobacteriaceae (n=51,352) and Pseudomonas aeruginosa (n=11,842) collected from hospitalized patients in 208 medical center laboratories from 40 countries from 2012 to 2015 for in vitro susceptibility to aztreonam-avibactam, aztreonam, and comparator antimicrobial agents using standard broth microdilution methodology. Avibactam was tested at a fixed concentration of 4 μg/ml in combination with two-fold dilutions of aztreonam. The concentration of aztreonam-avibactam and aztreonam inhibiting 90% of isolates (MIC90) was 0.12 and 64 μg/ml for all Enterobacteriaceae isolates; >99.9% of all isolates and 99.8% of meropenem-nonsusceptible isolates (n=1,498) were inhibited by aztreonam-avibactam at a concentration of ≤8 μg/ml. PCR and DNA sequencing identified 267 Enterobacteriaceae isolates positive for MBL genes (NDM, VIM, IMP); all Enterobacteriaceae carrying MBLs demonstrated aztreonam-avibactam MICs of ≤8 μg/ml and a MIC90 of 1 μg/ml. Against all P. aeruginosa isolates tested, the MIC90 of both aztreonam-avibactam and aztreonam was 32 μg/ml; against MBL-positive P. aeruginosa (n=452) MIC90 values for aztreonam-avibactam and aztreonam were 32 and 64 μg/ml, respectively. The current study demonstrated that aztreonam-avibactam possessed potent in vitro activity against a recent, sizeable global collection of Enterobacteriaceae clinical isolates, including isolates that were meropenem-non-susceptible, and against MBL-positive isolates of Enterobacteriaceae, for which there are few treatment options.