Background The aim of this study was to quantify mediators of neutrophilic inflammation within airway extracellular vesicles (EVs) of children treated for a cystic fibrosis (CF) pulmonary exacerbation (PEx). Methods EVs were isolated from stored sputum samples collected before and after antibiotic therapy for PEx between 2011–2013, and characterised by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Western blot analysis of EV protein extracts was used for EV canonical protein markers CD63, CD9, and flotillin-1 (FLOT1), as well as neutrophil elastase (NE), myeloperoxidase (MPO) and interleukin-8. EV content of NE and MPO was expressed as ratios of NE/FLOT1 and MPO/FLOT1 protein band densities. Results Sputum samples from 21 children aged 13.3 (range 8.0–17.0) years were analysed. NTA showed high concentrations of particles at the size of small EVs (50–200 nm), and typical EV morphology was confirmed by TEM. CD63, CD9 and FLOT1 was detectable in all samples. Median (IQR) NE/FLOT1 increased from 2.46 (1.68–5.25) before to 6.83 (3.89–8.89, p<0.001) after PEx therapy and MPO/FLOT1 from 2.30 (1.38–4.44) to 5.76 (3.45–6.94, p<0.01), while EV size remained unchanged. Improvement in lung function (ppFEV 1 ) with PEx therapy correlated with NE EV content (r=0.657, p=0.001). Conclusions Airways of children with CF contain EVs that carry NE and MPO as cargo. The lower NE and MPO content at the time of PEx compared to after therapy and the correlation with pulmonary function suggest both a functional role of EVs in CF airway inflammation and potential as a biomarker to monitor CF lung disease.