In this work, for the first time, we showed the successful utilization of enzymatic-based (bilirubin oxidase, BOx) air-breathing gas-diffusion (ABGD-BOx) cathode in membraneless single chamber microbial fuel cells (SCMFCs). At pH range of 7.2-7.5, the cathode open circuit potentials (OCPs) achieved were in the range of 490-535 mV (vs. sat. Ag/AgCl) with the anodic OCP being between −495 mV and −530 mV (vs. sat. Ag/AgCl). The highest overall open circuit voltage (OCV) of the SCMFCs (1.05 V) fed with phosphate buffer solution (PBS) and sodium acetate (NaOAc) was achieved after 12-24 hour of operation. Polarization curves of the ABGD-BOx cathode showed lower overpotential with an advantage of up to 250 mV compared to the traditional platinum Pt-based cathode, and possessed a great potential to replace the noble metal-based cathodes in MFCs. High power generation of 2 W m −2 (200 μW cm −2 , referred to cathode geometric surface area) was achieved in SCMFCs with the ABGD-BOx cathode, which was the highest power generation reported for SCMFCs with a volume greater than 100 mL.The Microbial fuel Cell (MFC) is a promising bio-electrochemical system capable of converting organic compounds (e.g. organic wastes) into useful electricity. It consists of two half-cells -the anode and the cathode -which are typically separated by an ion selective membrane. The anode electrode is immersed in a solution containing organic compounds, and the anodophillic bacteria, growing on the anode surface, oxidize the organic substances through their metabolic processes. At the cathode, oxygen is normally used as the final electron acceptor. In this case, the oxygen reacts with the electrons generated from the anode and along with the protons, released during the oxidation at the anode, produces water.To accelerate the cathodic oxygen reduction reaction (ORR), catalysts (e.g. noble metals, platinum) are quite often used in MFCs. Even though the efficiency of the platinum (Pt)-based cathodes has been reported to be high in acidic solutions, 1 it is actually significantly lower in the neutral/mildly alkaline solutions. 1 The cathode open circuit potential (OCP) achieved with Pt-based electrodes in MFCs at ambient temperatures and neutral media is relatively low (0.3-0.4 V vs sat. Ag/AgCl 2 ), compared to the theoretical ORR potential (∼0.62 V vs. sat. Ag/AgCl). This is an indication of high overpotentials for the ORR. Moreover, the direct contact of the catalyst with the solution in the MFCs, leads to cathode flooding and large ohmic losses, 2 which lowers the overall performance of MFCs.In order to reduce the MFC costs, enhance power generation, and prolong the lifetime of cathodic systems, extensive studies have been conducted to develop novel cost-effective metal-based 3-6 or nonmetal-based 7-10 catalysts. It has been found that the ORR kinetics of these metals/non-metal-based cathodes enhanced significantly at higher temperatures, 11 indicating that this type of reaction is hindered in MFCs operated at ambient temperature.Enzymes have recent...