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The bioelectrooxidation of ethanol was investigated in a fully enzymatic membraneless ethanol/O 2 biofuel cell assembly using hybrid bioanodes containing multi-walled carbon nanotube (MWCNT)-decorated gold metallic nanoparticles with either a pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) enzyme or a nicotinamide adenine dinucleotide (NAD + )-dependent ADH enzyme. The biofuel cell anode was prepared with the PQQ-dependent enzyme and designed using either a direct electron transfer (DET) architecture or via a mediated electron transfer (MET) configuration through a redox polymer, 1,1′-dimethylferrocene-modified linear polyethyleneimine (FcMe 2 -C 3 -LPEI). In the case of the bioanode containing the NAD + -dependent enzyme, only the mediated electron transfer mechanism was employed using an electropolymerized methylene green film to regenerate the NAD + cofactor. Regardless of the enzyme being employed at the anode, a bilirubin oxidase-based biocathode prepared within a DET architecture afforded efficient electrocatalytic oxygen reduction in an ethanol/O 2 biofuel cell. The power curves showed that DET-based bioanodes via the PQQ-dependent ADH still lack high current densities, whereas the MET architecture furnished maximum power density values as high as 226 ± 21 μW cm −2 . Considering the complete membraneless enzymatic biofuel cell with the NAD + -dependent ADH-based bioanode, power densities as high as 111 ± 14 μW cm −2 were obtained. This shows the advantage of PQQ-dependent ADH for membraneless ethanol/O 2 biofuel cell applications. [ABSTRACT FROM AUTHOR]