A better understanding of how anode and separator physical properties affect power production is needed to improve energy and power production by microbial fuel cells (MFCs). Oxygen crossover from the cathode can limit power production by bacteria on the anode when using closely spaced electrodes [separator electrode assembly (SEA)]. Thick graphite fiber brush anodes, as opposed to thin carbon cloth, and separators have previously been examined as methods to reduce the impact of oxygen crossover on power generation. We examined here whether the thickness of the anode could be an important factor in reducing the effect of oxygen crossover on power production, because bacteria deep in the electrode could better maintain anaerobic conditions. Carbon felt anodes with three different thicknesses were examined to see the effects of thicker anodes in two configurations: widely spaced electrodes and SEA. Power increased with anode thickness, with maximum power densities (604 mW/m 2, 0.32 cm; 764 mW/m2, 0.64 cm; and 1048 mW/m2, 1.27 cm), when widely spaced electrodes (4 cm) were used, where oxygen crossover does not affect power generation. Performance improved slightly using thicker anodes in the SEA configuration, but power was lower (maximum of 689 mW/m2) than with widely spaced electrodes, despite a reduction in ohmic resistance to 10 Ω (SEA) from 51-62 Ω (widely spaced electrodes). These results show that thicker anodes can work better than thinner anodes but only when the anodes are not adversely affected by proximity to the cathode. This suggests that reducing oxygen crossover and improving SEA MFC performance will require better separators. © 2012 American Chemical Society.