In recent years, pressure-assisted forward osmosis (PAFO) process has been studied to overcome the low water productivity in FO-RO hybrid seawater desalination. Our research group demonstrated a pilot-scale PAFO-RO plant for seawater desalination operations using secondary treated wastewater and natural seawater. A pretreatment stage of mesh tube filter media (MTF) and cartridge filter (CF) was implemented for a pilot-scale PAFO. In this study, the water quality and fouling index (SDI₁₅ and MFI_0.45) of the influent and treated water at each process stage of the plant were measured. For the influent of both wastewater and seawater feature, it showed linear correlations between the concentration of microbial ATP (i.e., an indicator of microbial activity) and MFI_0.45. After the PAFO was operated for 76 days, the fouling of the filtration media and the FO membrane was also analyzed. The observation of the fouled membranes by SEM showed that fouling was hardly observed on the side contacting with seawater, but severe bacterial biofilms were observed on the side contacting with wastewater. FT-IR results also confirmed protein-like substances on the membrane surface contacting with wastewater, indicating irreversible fouling. This study suggests that an advanced pretreatment process to remove bacteria and organic matter is essential for the protection of the FO membranes.