Respiratory disease is the main cause of morbidity and mortality in patients with cystic fibrosis (CF). In such patients chronic Pseudomonas aeruginosa infection is virtually impossible to eradicate using antibiotic therapy. Photodynamic antimicrobial chemotherapy (PACT) could be one potential alternative antimicrobial method. As photosensitisers could be delivered to the lungs of CF patients via inhalation, the current in vitro study investigated the potential use of PACT in the treatment of P. aeruginosa CF pulmonary infection. Delivery of red light (635 nm) and two photosensitisers (toluidine blue O (TBO) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP)) across artificial CF mucus was successfully achieved. Artificial CF mucus reduced the measured fluence of incident light in an almost exponential manner with increasing depth. The presence of dissolved photosensitisers also reduced light fluence. TMP diffused more efficiently across artificial CF mucus than TBO. However, receiver compartment concentrations of both drugs after 6 h were of the same order as those required to achieve high rates of kill (>99%) of P. aeruginosa isolates growing both planktonically and in biofilms. TMP required significantly higher concentrations (2.5 mg ml−1) than TBO to achieve high rates of kill (>99%) of P. aeruginosa isolates growing planktonically. Higher concentrations (5.0 mg ml−1) of both photosensitisers were required to achieve high rates of kill (>99%) of P. aeruginosa isolates growing in biofilms. When photosensitisers were prepared in artificial mucus, higher concentrations were required to achieve reasonably high kill rates (>80%) of P. aeruginosa (PAO1) growing both planktonically and in biofilm.