The mushroom industry in Asian countries has been progressing throughout the years. However, as mushroom production increases, the generation of spent mushroom substrates (SMS) also increases, resulting in pollution of land, water, and air. These SMS contain a significant amount of biomass composed of cellulose, hemicellulose, lignin, and phenolic groups, which can be further degraded using microbial fuel cells (MFCs). The most popular type of mushroom produced in the Philippines is the oyster mushroom (Pleurotus ostreatus). P. ostreatus, is a white-rot basidiomycete fungus producing various extracellular enzymes including Mn-peroxidase, lignin peroxidase, and laccase. These enzymes have the potential to degrade agro-wastes while synergistically generating electricity. Specifically, laccase, acting as phenol-degrading peroxidase, can decompose SMS while simultaneously serving as a catalyst for the electrochemical flow within the MFC. With this, the utilization of the extracellular activities of laccase derived from P. ostreatus in MFCs with varying salinity of electrolytes was investigated. The current, voltage, and power density of the MFCs were recorded in 12-hour intervals for 10 days using a multimeter. The laccase activity was then measured at days 0, 5, and 10 through an enzymatic assay by colorimetry test using guaiacol as substrate. The MFCs generated a maximum power density of 480.201 mW/cm2, and a maximum current density of 0.002 A/cm2, along with the maximum observed laccase activity of 229.970 U/L on Day 10. These results suggest that incorporating live P. ostreatus inoculates could be a promising approach to enhance the capacity of MFCs for energy production and waste degradation.