A factorial design was used to optimize the extraction of polycyclic aromatic hydrocarbons (PAHs) from mosses, plants used as biomonitors of air pollution. The analytical procedure consists of pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) cleanup, in association with analysis by high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). For method development, homogeneous samples were prepared with large quantities of the mosses Isothecium myosuroides Brid. and Hypnum cupressiforme Hedw., collected from a Spanish Nature Reserve. A factorial design was used to identify the optimal PLE operational conditions: 2 static cycles of 5 min at 80°C. The analytical procedure performed with PLE showed similar recoveries (∼70%) and total PAH concentrations (∼200 ng g-1) as found using Soxtec extraction, with the advantage of reducing solvent consumption by 3 (30 mL against 100 mL per sample), and taking a fifth of the time (24 samples extracted automatically in 8 h against 2 samples in 3.5 h). The performance of SPE normal phases (NH2, Florisil®, silica and activated aluminium) generally used for organic matrix cleanup was also compared. Florisil® appeared to be the most selective phase and ensured the highest PAH recoveries. The optimal analytical procedure was validated with a reference material and applied to moss samples from a remote Spanish site in order to determine spatial and inter-species variability.