Antimicrobial resistance poses a global threat to public health. Of great concern areAcinetobacter baumannii,Pseudomonas aeruginosaand Enterobacterales with resistance to carbapenems or third-generation cephalosporins. The aim of the present study was to investigate the in vitro activity of the novel siderophore cephaloporin cefiderocol (CID) and four comparator β-lactam-β-lactamase-inhibitor combinations and to give insights into the genetic background of CID-resistant isolates. In total, 301 clinical Enterobacterales and non-fermenting bacterial isolates were selected for this study, including randomly chosen isolates (set I, n = 195) and challenge isolates (set II, n = 106; enriched with ESBL and carbapenemase producers, as well as colistin-resistant isolates). Isolates displayed CID MIC50/90values of 0.12/0.5 mg/L (set I) and 0.5/1 mg/L (set II). Overall, the CID activity was superior to the comparators againstA. baumannii,Stenotrophomonas maltophiliaand set II isolates ofP. aeruginosa. There were eight CID-resistant isolates detected (MIC > 2 mg/L):A. baumannii(n = 1),E. cloacaecomplex (n = 5) andP. aeruginosa(n = 2). Sequencing analyses of these isolates detected the acquired β-lactamase (bla) genesblaNDM-1,blaSHV-12and naturally occurringblaOXA-396,blaACT-type andblaCMH-3. In conclusion, CID revealed potent activity against clinically relevant organisms of multidrug-resistant Enterobacterales and non-fermenters.