Abstract The effect of the diffusion layer on the performance and mass transport in a direct methanol fuel cell at ambient conditions is reported. Carbon cloths with variable Teflon contents and pore sizes, carbon paper and a metal wire cloth were investigated. Membrane-electrode-assemblies (MEAs) for direct methanol fuel cells (DMFCs) prepared after an in-house receipt are used, giving reproducible results after a pre-treatment involving polarisation with hydrogen and air. Long-term effects and methanol crossover were also briefly investigated. Adding Teflon to the diffusion layer leads to better gas transport, as gas and liquid transport takes place in different paths. Thus, the fuel cell power output is more stable. The same effect was seen with increasing pore size. Carbon paper is found not suitable as a diffusion layer for low temperature DMFC. The metal wire cloth yielded best performance giving 15.8 mW cm−2.