© 2014 Elsevier Ltd. Separators are used to protect cathodes from biofouling and to avoid electrode short-circuiting, but they can adversely affect microbial fuel cell (MFC) performance. A spray method was used to apply a polyvinyl alcohol (PVA) separator to the cathode. Power densities were unaffected by the PVA separator (339 ± 29 mW/m2), compared to a control lacking a separator in a low conductivity solution (1mS/cm) similar to wastewater. Power was reduced with separators in solutions typical of laboratory tests (7-13 mS/cm), compared to separatorless controls. The PVA separator produced more power in a separator assembly (SEA) configuration (444 ± 8 mW/m2) in the 1mS/cm solution, but power was reduced if a PVA or wipe separator was used in higher conductivity solutions with either Pt or activated carbon catalysts. Spray and cast PVA separators performed similarly, but the spray method is preferred as it was easier to apply and use.