This study describes on-line monitoring and parameter estimation during Microbial Fuel Cell operation with a pulse-width modulated connection of the external resistor (R-PWM mode) at low and high frequencies. Analysis of the output voltage profiles acquired during R-PWM tests showed the presence of slow and fast dynamic components, which can be described by an equivalent circuit model suitable for process monitoring and control applications. To demonstrate the proposed monitoring and parameter estimation procedure, the MFC was operated at several influent concentrations of acetate (carbon source) and an on-line parameter estimation procedure was used for estimating internal resistance and internal capacitance. Furthermore, these parameters were re-estimated at the end of each test yielding similar results. The proposed on-line procedure can be used for real-time process optimization.