The novel siderophore cephalosporin cefiderocol (S-649266) with potent activity against Gram-negative pathogens was recently developed (Shionogi & Co., Ltd.). Here, we evaluated the activity of this new molecule and comparators against a collection of previously characterized Gram-negative isolates using broth microdilution panels. A total of 753 clinical multidrug-resistant Gram-negative isolates collected from hospitals worldwide were tested against cefiderocol and antibiotic comparators (ceftolozane-tazobactam [CT], meropenem [MEM], ceftazidime [CAZ], ceftazidime-avibactam [CZA], colistin [CST], aztreonam [ATM], amikacin [AMK], ciprofloxacin [CIP], cefepime [FEP], and tigecycline [TGC]) for their susceptibility. The collection included Escherichia coli (n = 164), Klebsiella pneumoniae (n = 298), Enterobacter sp. (n = 159), Pseudomonas aeruginosa (n = 45), and Acinetobacter baumannii (n = 87). Resistance mechanisms included producers of carbapenemases and extended-spectrum β-lactamases (ESBLs). In addition, a series of colistin-resistant enterobacterial isolates (n = 74), including 15 MCR-1 producers, were tested. The MIC90 of cefiderocol was 2 mg/L, while those of comparative drugs were >64 mg/L for CT, MEM, CAZ, CZA, and AMK, >32 mg/L for ATM, >16 mg/L for FEP, 8 mg/L for CST, and 2 mg/L for TGC. The MIC50 of cefiderocol was 0.5 mg/L, while those of other drugs were >64 mg/L for CAZ, 64 mg/L for CT, >32 mg/L for ATM, >16 mg/L for FEP, 8 mg/L for MEM and AMK, >4 mg/L for CIP, 1 mg/L for CZA, 0.5 mg/L for TGC, and <0.5 mg/L for CST. Only 20 out of 753 strains showed MIC values of cefiderocol ≥8 μg/mL. Compared to the other drugs tested, cefiderocol was more active, with the exception of colistin and tigecycline showing equivalent activity against certain subgroups of bacteria.