Separators are needed in microbial fuel cells (MFCs) to reduce electrode spacing and preventing electrode short circuiting. The use of nylon and glass fiber filter separators in single-chamber, air-cathode MFCs was examined for their effect on performance. Larger pore nylon mesh were used that had regular mesh weaves with pores ranging from 10 to 160 μm, while smaller pore-size nylon filters (0.2-0.45 μm) and glass fiber filters (0.7-2.0 μm) had a more random structure. The pore size of both types of nylon filters had a direct and predictable effect on power production, with power increasing from 443 ± 27 to 650 ± 7 mW m-2 for pore sizes of 0.2 and 0.45 μm, and from 769 ± 65 to 941 ± 47 mW m-2 for 10 to 160 μm. In contrast, changes in pore sizes of the glass fiber filters resulted in a relatively narrow change in power (732 ± 48 to 779 ± 43 mW m-2) for pore sizes of 0.7 to 2 μm. An ideal separator should increase both power density and Coulombic efficiency (CE). However, CEs measured for the different separators were inversely correlated with power production, demonstrating that materials which reduced the oxygen diffusion into the reactor also hindered proton transport to the cathode, reducing power production through increased internal resistance. Our results highlight the need to develop separators that control oxygen transfer and facilitate proton transfer to the cathode. © 2010 The Royal Society of Chemistry.