Chronic respiratory infection caused by Pseudomonas aeruginosa is the main driver of morbidity and mortality in cystic fibrosis (CF) patients. The development of resistance to all available antibiotics is a frequent outcome of these infections. The present study aimed to evaluate the activity of the new cephalosporin CXA-101 (FR264205) against a collection of 100 isolates obtained from 50 CF patients from two Spanish hospitals. The collection included the first (early) and the last (late) available isolate from each patient (average interval 68 ± 39 months). The MIC50 and MIC90 of CXA-101 were 0.5 and 2 mg/L and the geometric mean MIC was 0.7 mg/L; the MICs for 95% of the isolates were ≤8 mg/L (tentative breakpoint). Only meropenem yielded comparable results, although the MIC90 of this antibiotic was significantly higher (8 mg/L). CXA-101 showed conserved activity against a high proportion of isolates resistant to each of the antibiotics tested (ceftazidime, cefepime, piperacillin-tazobactam, imipenem, meropenem, levofloxacin and tobramycin), with MIC50 values of 1-2 mg/L. Moreover, CXA-101 retained good activity against multidrug-resistant strains, with MIC50 and MIC90 values of 2 and 16 mg/L. CXA-101 was also active against late CF isolates (the MIC for 96% was ≤8 mg/L); it was the only antibiotic tested to which a similar percentage of early and late isolates was susceptible. These results show that, despite a slight increase in MICs, major cross-resistance to CXA-101 did not develop during treatment of CF patients with the currently available antipseudomonal agents. Therefore, CXA-101 is envisaged as a valuable alternative for the treatment of chronic respiratory infection caused by P. aeruginosa in CF patients.