Zidebactam is a β-lactam enhancer antibiotic with a dual mechanism of action involving binding to Gram-negative PBP2 and β-lactamase inhibition. Cefepime combined with zidebactam (WCK 5222) is under clinical development for treatment of Gram-negative infections. To evaluate the in vitro activities of cefepime and zidebactam separately and combined at 1:1 and 2:1 ratios when tested against Gram-negative organisms producing the most clinically relevant β-lactamase types. β-Lactamase-producing (193) and WT (71) isolates were tested for susceptibility by broth microdilution method against cefepime/zidebactam, cefepime and zidebactam. Cefepime/zidebactam (1:1) was very active against Enterobacteriaceae producing CTX-M-15 (21; MIC 50/90 0.25/1 mg/L), SHV (20; MIC 50/90 0.12/0.25 mg/L), other ESBLs (20, including GES-18, OXA-1/30 and OXY-, PER-, TEM- and VEB-like; MIC 50/90 0.25/1 mg/L), plasmidic AmpC (10; MIC 50/90  ≤0.06/≤0.06 mg/L), derepressed AmpC (23; MIC 50/90 0.12/0.5 mg/L), KPC (35; MIC 50/90 0.25/1 mg/L) and metallo-β-lactamases (MBLs; 20 including VIM, IMP and NDM; MIC 50/90 0.5/8 mg/L). Cefepime/zidebactam (1:1) was also active against Pseudomonas aeruginosa with overexpression of AmpC (21; MIC 50/90 4/8 mg/L) and MBLs [12 (VIM and IMP); MIC 50/90 4/8 mg/L]. Zidebactam alone exhibited potent in vitro activity against some Enterobacteriaceae and P. aeruginosa , including β-lactamase-producing strains. Cefepime/zidebactam MIC values were lower than those of each agent tested alone for many β-lactamase-producing strains, indicating synergy. Cefepime/zidebactam showed moderate activity against OXA-23/24/58-producing Acinetobacter baumannii [MIC 50/90 32 mg/L (1:1)]. Cefepime/zidebactam showed potent activities against Enterobacteriaceae and P. aeruginosa producing various clinically relevant β-lactamases, including ESBLs, KPCs, AmpC and MBLs for which limited treatment options are currently available.