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 mu W 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. (C) 2013 The Electrochemical Society. All rights reserved.