This work highlights the importance of developing technologies for full ethanol oxidation, to obtain high energy production rates. We have carried out experimental and theoretical investigations to unveil the action modes of the enzymes ADH and AldDH toward ethanol, as well as the influence of the organic catalyst TEMPO on the process. Ethanol interacted better with the complex ADH/TEMPO. The energy gap (HOMO – LUMO) for the enzymes complexed with TEMPO was lower. Ethanol showed more stabilizing interaction energies when it was docked in the active site of the enzymes complexed with TEMPO, which indicated the relevance of this organic catalyst. AldDH alone did not oxidize ethanol, but this changed when this enzyme was inserted into a biofuel cell (BFC) system containing TEMPO—the electron flow seemed to be optimized in the presence of this catalyst. On the basis of our experimental results, the specific activity of ethanol oxidation through ADH was much higher as compared to acetaldehyde oxidation through AldDH. This study opens new possibilities for applying this multifunctional hybrid system for optimal energy harvesting from biofuels.