Chronic airway colonisation by Pseudomonas aeruginosa, a hallmark of cystic fibrosis (CF) lung disease, is associated with increased morbidity and mortality and despite aggressive antibiotic treatment, P. aeruginosa is able to persist in CF airways. In vitro antibiotic susceptibility assays are poor predictors of antibiotic efficacy to treat respiratory tract infections in the CF patient population and the selection of the antibiotic(s) is often made on an empirical base. In the current review, we discuss the factors that are responsible for the discrepancies between antibiotic activity in vitro and clinical efficacy in vivo. We describe how the CF lung microenvironment, shaped by host factors (such as iron, mucus, immune mediators and oxygen availability) and the microbiota, influences antibiotic activity and varies widely between patients. A better understanding of the CF microenvironment and population diversity may thus help improve in vitro antibiotic susceptibility testing and clinical decision making, in turn increasing the success rate of antibiotic treatment.