The direct methanol fuel cell (DMFC) is a promising power source for micro- and various portable electronic devices (mobile phones, PDAs, laptops, and multimedia equipment) with the advantages of easy fuel storage, no need for humidification, and simple design. However, a number of issues need to be resolved before DMFC commercialization, such as the methanol crossover and water crossover, which must be minimized in portable DMFCs. In the present work, a detailed experimental study on the performance of an “in-house” developed DMFC with of active membrane area, working near the ambient conditions is described. The influence on the DMFC performance of the methanol concentration in the fuel feed solution and of both anode and cathode flowrates was studied. Tailored membrane electrode assemblies (MEAs) were designed in order to select optimal working conditions. Different structures and combinations of gas diffusion layers (GDLs) were tested. Under the operating conditions studied it was shown that, as expected, the cell performance significantly increases with the introduction of gas diffusion layers and that carbon cloth is more efficient than carbon paper both for the anode and cathode GDLs. The results reported allow the setup of tailored MEAs enabling the cell operation at high methanol concentrations (high power densities) without sacrificing performance (i.e., achieving low methanol crossover values). The influence of the different parameters on the cell performance is explained under the light of the predictions from a previously developed one-dimensional model, coupling heat and mass transfer effects. The main gain of this work is to report DMFC detailed experimental data at near ambient temperature which are insufficient in literature. This operating condition is of special interest in portable applications.