The marine algae Chaetoceros contains hexadecatrienoic acid, which suppresses methanogen development and improves the coulombic efficiency (CE) of microbial fuel cells (MFC). To inhibit the methanogens, optimum concentration of marine algae should be added to the anaerobic sludge to enhance the performance of MFC. A varying concentration of Chaetoceros ranging from 1 to 20 mg/mL was carried out for pretreatment of an anaerobic-mix consortium to suppress methanogens. MFC inoculated with pretreated anaerobic sludge with 10 mg/mL Chaetoceros showed a maximum power density of 21.62 W/m3 and a maximum CE of 37.25%, which was considerably higher than the treatment with other concentrations. At 10 mg/mL concentration, Tafel analysis of the anode in the MFC showed a higher exchange current density of 66.35 mA/m2 and a lower charge transfer resistance of 0.97 Ω.m2, revealing higher bio-electrochemical activity. The performance of MFC improved when the concentration of Chaetoceros was increased up to 10 mg/mL, but then began to decline as the concentration increased further. Thus, the optimum dose of Chaetoceros to be added in the mix-anaerobic consortium to optimize the power performance of MFC was determined, which can be carried out in scaled-up MFCs.