ABSTRACTIn this study, we aimed to comparatively evaluate thein vitroactivity of cefiderocol versus other antimicrobials against a well-characterized collection of metallo-beta-lactamase (MBL)-producing Gram-negative bacilli (MBL-GNB) isolates from hospitals in Andalusia, Spain. We recovered 232 MBL-GNB from Andalusian hospitals, including 160 Enterobacterales and 72 nonfermenting Gram-negative bacilli belonging to 44 different clones (2015 to 2020). Cefiderocol and comparator MICs were determined with commercial methods (UMIC [Bruker] and EUMDROXF [Sensititre; Thermo Fisher], respectively). EUCAST breakpoints were used for all antimicrobials tested, and CLSI also was used for cefiderocol. Control strains used wereE. coliATCC 25922 andPseudomonas aeruginosaATCC 27853. Cefiderocol showed potentin vitroactivity against isolates tested, regardless of breakpoint (susceptibility rates, 85.3% for EUCAST versus 96.6% for CLSI,P <0.001). MIC ranges for Enterobacterales and nonfermenting Gram-negative bacilli (NF-GNB) were ≤0.03 to 1 mg/L and 0.06 to 2 (IMP), 0.06 to 8 mg/L and 0.06 to 16 (VIM), 0.25 to 16 mg/L and 2 to 16 mg/L (NDM), respectively, and 0.25 to 8 mg/L for double MBL-producing Enterobacterales. By species, all cefiderocol-susceptible rates were over 90%, exceptKlebsiella oxytoca,Enterobacter cloacae,Escherichia coli, andAcinetobacterspp. Significant differences were observed comparing resistant isolates between Enterobacterales and NF-GNB by EUCAST (19.4% versus 4.2%,P <0.01), but not by CLSI (4.4% versus 1.4%,P =0.2). Cefiderocol was the most active antimicrobial tested. Cefiderocol showed excellentin vitroactivity against MBL-GNB, especially NF-GNB; almost all isolates resistant to comparators were susceptible.IMPORTANCEThis article demonstrates the efficacy of cefiderocol against a large collection of well-characterized metallo-beta-lactamase-producing isolates, some of them even producing double carbapenemases. Furthermore, cefiderocol activity is compared to other novel broad-spectrum antimicrobials with activity against carbapenemases.