Edible mushroom laccase is a fascinating extracellular enzyme owing to its simple operation. This research examined (a) the electrochemical properties of a single-chamber microbial fuel cell with two edible mushrooms (Pleurotus pulmonarius and Coprinus fimetarrius), and (b) the laccase activity of edible mushrooms grown in spent mushrooms as a low-cost substrate. The results revealed that the single-chamber MFC with P. pulmonarius achieved a maximum open-circuit voltage of 940.00±10.00 mV, a maximum current density of 97.14±2.86 mA/m², and a PD of 13.22±0.78 mW/m². When P. pulmonarius was cultured in a sawdust substrate without chemical addition, laccase activity of 155.17±0.10 U/mL was obtained. There has been no previous report of electricity generation from a membrane-less single-chamber MFC using P. pulmonarius mycelium cultured on sawdust substrate as a whole-cell cathode catalyst.