Background: Methods: We collected mucus from primary non-CF and CF human bronchial epithelial cells stimulated with interleukin (IL)-4, IL-1β, pyocyanin (PYO) or co-infected with PsA. Paired sputum samples from people with CF before and after elexacaftor/tezacaftor/ivacaftor (ETI) treatment were also utilised. Af infection was then performed directly on each mucus sample and imaged with microscopy. Images were analysed by ImageJ particle tracking. Results: Higher concentrations of CF and non-CF mucus promoted more Af growth. Af germinated more in IL-4-stimulated CF mucus than CF mucus alone as well as IL-4-stimulated non-CF mucus, even when controlling for per cent solid content. PYO exposure showed increased Af growth in CF and non-CF mucus, while co-infection of CF mucus with PsA and tobramycin treatment restored Af growth in both conditions. Paired sputum samples from people with CF showed more Af growth pre-ETI than post-ETI even when controlling for per cent solid content. Conclusion: Af growth is reduced by mucus dilution, promoted by specific inflammatory cytokines and infection, and is influenced by CFTR function. CF mucus shows pathological differences that promotes Af growth, suggesting an intrinsic defect in this population.