The role of the electroactive surface area (EASA) and of the non-electroactive surface area (nEASA) was studied to better understand electroactive biofilm’s (EAB) growth and performance in four different systems. Those systems consisted in four 1L glass bottles filled with mineral medium and substrates, a stainless-steel cathode and a bioanode. Four different types of bioanode were assembled in order to study the EASA and nEASA role. A potentiostat controlled the anodic potential, which was fixed in every system at + 0.2 V vs SHE (standard hydrogen electrode). To measure the EASA of every system, cyclic voltammetries (CVs) were carried out at different scan rates. Comparing them with the one obtained with a reference system, each EASA is easily calculated. The nEASA, instead, was measured calculating the geometric volume. The obtained results demonstrate the fundamental role of the EASA and, moreover, the necessity to reduce as much as possible the nEASA in order to enhance the performance.