Next-generation pressure-retarded osmosis (PRO) approaches aim to harness the energy potential of streams with high salinity differences, such as wastewater effluent and seawater desalination plant brine. In this study, we evaluated biofouling propensity in PRO. Bench-scale experiments were carried out for 24 h using a model wastewater effluent feed solution and simulated seawater desalination brine pressurized to 24 bar. For biofouling tests, wastewater effluent was inoculated with Pseudomonas aeruginosa and artificial seawater desalination plant brine draw solution was seeded with Pseudoalteromonas atlantica. Our results indicate that biological growth in the feed wastewater stream channel severely fouled both the membrane support layer and feed spacer, resulting in ∼50% water flux decline. We also observed an increase in the pumping pressure required to force water through the spacer-filled feed channel, with pressure drop increasing from 6.4±0.8 bar m(-1) to 15.1±2.6 bar m(-1) due to spacer blockage from the developing biofilm. Neither the water flux decline nor the increased pressure drop in the feed channel could be reversed using a pressure-aided osmotic backwash. In contrast, biofouling in the seawater brine draw channel was negligible. Overall, the reduced performance due to water flux decline and increased pumping energy requirements from spacer blockage highlight the serious challenges of using high fouling potential feed sources in PRO, such as secondary wastewater effluent. We conclude that PRO power generation using wastewater effluent and seawater desalination plant brine may become possible only with rigorous pretreatment or new spacer and membrane designs.