Natural eggshell membrane (ESM) is proposed as a separator to enhance electricity recovery in air-cathode microbial fuel cells (MFCs). The unique fibrous structure and surface chemistry of ESM can mitigate substrate and oxygen crossover, and biofouling of the cathode, achieving a remarkable coulombic efficiency of 67.14–95.03%, depending on the current density. This study demonstrates an effective, simple and green route for recovering electricity from organic matter in MFCs.