Microbial fuel cells (MFCs) have been receiving an increased attention over the last years due to their potential to combat two global problems: waste pollution and energy demand. Additionally, when a wastewater is used, MFCs can perform its treatment while recovering energy, leading to the possibility of energy-producing wastewater treatment plants, offsetting their operational costs. However, to overcome their current limitations (lower power outputs and higher costs), a clear understanding of the effect of operation and design parameters on its overall performance is mandatory. Therefore, the goal of this work was to evaluate the effect of operating conditions - batch cycle and yeast extract concentration, and design parameters - anode electrode area, membrane thickness and active area, on the overall performance of a single chamber MFC. The MFC operated with a pure culture of Lactobacillus pentosus and a synthetic wastewater based on a real dairy industry effluent. The overall performance was evaluated through the power output and the COD removal rate. Additionally, the biofilm formed at the anode electrode was characterized in terms of biomass, proteins and polysaccharides content. For the conditions used in this work, a maximum power density of 5.04 ± 0.39 mW/m2 was achieved with an anode electrode area of 61 cm2, a batch cycle of 48 h, 50 mg/L of yeast extract and a Nafion 212 membrane with an active area of 25 cm2. The different conditions tested had a clear effect on the MFC energy production and biofilm characteristics, but not on the ability of L. pentosus to treat the dairy wastewater. The COD removal rates were in the range between 42% and 58%, for all the conditions tested.