Electrochemical impedance spectroscopy has received significant attention recently as a method to measure electrochemical parameters of Geobacter sulfurreducens biofilms. Here, we use electrochemical impedance spectroscopy to demonstrate the effect of mass transfer processes on electron transfer by G. sulfurreducens biofilms grown in situ on an electrode that was subsequently rotated. By rotating the biofilms up to 530 rpm, we could control the microscale gradients formed inside G. sulfurreducens biofilms. A 24% increase above a baseline of 82 µA could be achieved with a rotation rate of 530 rpm. By comparison, we observed a 340% increase using a soluble redox mediator (ferrocyanide) limited by mass transfer. Control of mass transfer processes was also used to quantify the change in biofilm impedance during the transition from turnover to non-turnover. We found that only one element of the biofilm impedance, the interfacial resistance, changed significantly from 900 to 4,200 Ω under turnover and non-turnover conditions, respectively. We ascribed this change to the electron transfer resistance overcome by the biofilm metabolism and estimate this value as 3,300 Ω. Additionally, under non-turnover, the biofilm impedance developed pseudocapacitive behavior indicative of bound redox mediators. Pseudocapacitance of the biofilm was estimated at 740 µF and was unresponsive to rotation of the electrode. The increase in electron transfer resistance and pseudocapacitive behavior under non-turnover could be used as indicators of acetate limitations inside G. sulfurreducens biofilms. Biotechnol. Bioeng. 2014;111: 285–294. © 2013 Wiley Periodicals, Inc.