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In passive direct ethanol fuel cells (DEFCs), the micro-porous layer (MPL) is a vital component of the membrane electrode assembly (MEA), facilitating gas-liquid mass transport and improving electronic conductivity. The conducted study involved preparing various carbon materials for the cathode MPL, including Ketjen Black (KB), activated carbon (AC) from Durian shells, and a 15% weight mixture of AC and KB (AC15%). Characterization of the activated carbon was carried out using nitrogen adsorption-desorption isotherm analysis. Additionally, various electrochemical techniques, including cell polarization, electrochemical impedance spectroscopy (EIS), anode half-cell polarization, and anode EIS, were conducted to examine the effects of the cathode MPLs on cell performance. The results indicated that the cell with the conventional KB cathode MPL displayed the highest performance, whereas the AC15% and AC cathode MPLs showed relatively lower performances, respectively. The AC cathode MPL in the cell encountered challenges, such as decreased pore volume, increased micropores, and a hydrophobic electrode nature, leading to reduced gas transport resulting in poor cell performance. In contrast, the AC15% cathode MPL, which combined AC and KB in the electrode, achieved an appropriate micropore and mesopore balance. However, performance did not improve due to a heterogeneous contact surface between the cathode catalyst layer and the cathode MPL, resulting in higher ohmic resistance. Incorporating biomass-based materials into the electrode presents an interesting possibility due to the utilization of cheap and readily available precursors, as well as the ability to tailor morphology. Conducting a systematic study of durian shell activated carbons would reveal improved properties of the carbon material suitable for implementing in the MPL of passive DEFCs.