Background: The management of chronic rhinosinusitis (CRS) in patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) is still a challenge. At our institution we have used gentamycin nasal spray, extemporaneously produced, for prophylactic treatment of moderate-to-severe CRS. The aim of this study was to investigate the gentamycin susceptibility of bacteria in sputum samples in CF and PCD patients treated for CRS. Methodology: Patients with CF and PCD who were prescribed gentamycin nasal spray for CRS and had sputum bacterial cultures taken pre-treatment and followed-up at least once after ≥6 months were retrospectively included. Microbiological data were descriptively analysed in terms of bacterial species and resistance to gentamycin. Results: A case series of 17 CF and 12 PCD patients passed the inclusion criteria. Of those cases, three (18%) CF patients and one (8%) PCD patient developed resistance to gentamycin during treatment with gentamycin nasal spray. In all four cases, the resistant bacterial isolates were <i>P. aeruginosa</i>. Additionally, two CF patients already had <i>P. aeruginosa </i> isolates resistant to gentamycin in the pre-treatment culture. In further two CF patients, the multi-resistant <i>Burgdorferi cepacia </i>complex, including gentamycin resistance, was identified. <i>P. aeruginosa </i> and <i>S. aureus </i> in CF and <i>P. aeruginosa</i> and <i>H. influenza </i> in PCD were the predominant bacterial species. Conclusions: The study showed that there was moderate incidence of gentamycin resistance in CF and PCD patients at our institution. However, further prospective studies are needed to confirm the outcomes.