We describe nanostructured electrode platforms composed of multiwalled carbon nanotube/glassy carbon electrodes (MWCNT/GCEs) with entrapped nitroaromatic compounds such as 2-nitrofluorene (2-NF), 2-nitro-9-fluorenone (2-NFN), 2,7-dinitrofluorene (2,7-NF) and 2,7-dinitro-9-fluorenone (2,7-NFN). All of the above nitroaromatic compounds were tested as precursors for the production of redox mediator couples useful for NADH electrocatalysis. In this communication, we reveal the effect of the substituents on both the π-π stacking interaction strength and the electrocatalysis of NADH. The results show that the nitro group plays a triple role: it acts as a precursor of the mediator redox couple for NADH electrocatalysis; it increases the π-π stacking interaction with the electrode; and it acts as an electron acceptor substituent that promotes the electrocatalysis of NADH.