For about 20 years, microbial fuel cells (MFCs) are an emerging technology that has gained attention for its new wastewater treatment and energy generation, especially its ability to convert chemical energy from a broad range of substances into electricity. However, MFC has not been widely commercialized due to low efficiency. Studies have shown that substrate loading is an important factor in scaling up. Therefore, this study investigates the effect of substrate type and concentration on honeycomb MFCs (HCMFCs). The effect of different concentrations ranging from 10 to 40 mM of lactate and acetate (1:1 ratio) substrates was investigated. Power efficiency was analyzed using polarization and power density curves. Results showed that the performance of MFCs and biofilm formation is affected by the substrates. Scanning electron microscopy showed some changes in biofilm formation. Mixing lactate and acetate at 30 mM gave the best performance with a power density of 956.75 mW m−2 and chemical oxygen demand removal of 87.8%. Furthermore, effective substrate degradation, having COD removal of 91.4%, was observed with acetate.