Pseudomonas aeruginosa is the principle pathogen implicated in progressive and recurrent respiratory infections in cystic fibrosis. Recently, impaired serum-mediated killing of P. aeruginosa was associated with increased severity of respiratory infections in patients with bronchiectasis. This inhibition was mediated by high titres of O-antigen-specific IgG2 antibodies. These ‘inhibitory antibodies’ were present in 20% of patients chronically infected with P. aeruginosa. Patients with these antibodies have been successfully treated with plasmapheresis. Here, we investigated the prevalence and mechanisms behind ‘inhibitory antibodies’ in patients with CF and P. aeruginosa infection without cognate isolates. IgG2 titres were measured in 75 serum samples obtained from patients with CF against eight P. aeruginosa serotypes. To confirm the inhibitory capacity of serum, serum bactericidal assays were performed. We found that 24 of 75 patients had serum that inhibited healthy control killing of P. aeruginosa. Interestingly, in a small number of patients with low IgG2 titres, increased antibody affinity appeared to mediate inhibition of killing. Moreover, in two patients with low IgG2 titres but inhibitory serum, we found that high titres of O-antigen specific IgA also inhibited killing of P. aeruginosa. Thus, inhibitory antibodies are highly prevalent (33%) in patients with CF and P. aeruginosa. Both titre and affinity of these antibodies is important for the inhibition. IgA specific for O-antigen can also inhibit serum-mediated killing even in the absence of IgG2. Thus, diagnostic screens for ‘inhibitory antibodies’ will need to account for titre, affinity and multiple isotypes binding to P. aeruginosa O-antigen.