The incidence of antimicrobial resistance in Europe is rising. Cefiderocol is approved in Europe for treatment of aerobic Gram-negative bacterial (GNB) infections in adults with limited treatment options. We report the in vitro activity of cefiderocol versus comparators against GNB clinical isolates from Spain. MICs were determined by broth microdilution according to International Organization for Standardization guidelines. Cefiderocol was tested using iron-depleted cation-adjusted Mueller–Hinton broth. Susceptibility rates were based on EUCAST breakpoints; if a species-specific breakpoint was unavailable, pharmacokinetic/pharmacodynamic breakpoints were used. Of 2303 isolates 1502 (65.2%) Enterobacterales and 801 (34.8%) non-fermenters, 2260 (98.1%) were susceptible to cefiderocol compared with 80.8–86.9% for comparators. By infection source, susceptibility to cefiderocol ranged from 97.3% (721/741) in isolates from patients with nosocomial pneumonia to 98.9% (349/353) in bloodstream infection isolates and was greater than susceptibility to comparators (70.7–93.6% across infection sources). Overall, 368/2303 isolates (16.0%) were meropenem-resistant. A high proportion of meropenem-resistant Acinetobacter baumannii 169/175 (96.6%) and Pseudomonas aeruginosa 48/50 (96.0%) were cefiderocol-susceptible, similar to colistin 169/175 (96.6%) and 47/50 (94.0%), respectively but higher than ceftazidime/avibactam 26/175 (14.9%) and 20/50 (40.0%), respectively and ceftolozane/tazobactam 17/175 (9.7%) and 25/50 (50.0%), respectively. All meropenem-resistant Stenotrophomonas maltophilia isolates 120/120 (100%) were cefiderocol-susceptible, including one trimethoprim/sulfamethoxazole-resistant isolate, with fewer susceptible to colistin 86/120 (71.7%), ceftazidime/avibactam 42/120 (35.0%) and ceftolozane/tazobactam 35/120 (29.2%). A high proportion of clinical isolates from Spain, representing a wide range of pathogens across multiple infection sources, were susceptible to cefiderocol. Cefiderocol retained activity against meropenem-resistant isolates. Display omitted