ABSTRACTSeparator is important to prevent bio-contamination of the catalyst layer of air-diffusion cathode microbial fuel cells (MFCs). Mixed cellulose ester filter (MCEF) was examined as a separator for an air-cathode MFC in the present study. The MCEF-MFC produced a maximum power density of 780.7 ± 18.7 mW/m2, which was comparable to 770.9 ± 35.9 mW/m2 of MFC with Nafion membrane (NFM) as a separator. Long-term examination demonstrated a more stable performance of the MCEF-MFC than NFM-MFC. After 25 cycles, the maximum voltage of the MCEF-MFC decreased by only 1.3% from 425.1 ± 4.3 mV (initial 5 cycles) to 419.5 ± 2.3 mV (last 5 cycles). However, it was decreased by 9.1% from 424.8 ± 5.7 to 386 ± 2.5 mV for the NFM-MFC. The coulombic efficiency (CE) of the MCEF-MFC did not change (from 3.11 ± 0.09% to 3.13 ± 0.02%), while it decreased by 9.12% from 3.18 ± 0.04% to 2.89 ± 0.02% for the NFM-MFC. The MCEF separator was with less biofouling than the NFM separator over 60 days’ operation, which might be the reason for the more table long-term performance of the MCEF-MFC. The results demonstrated that MCEF was feasible as a separator to set up good-performing and cost-effective air-diffusion cathode MFC.