Anode modification was explored as an approach to enhance the startup and improve the performance of microbial fuel cells (MFCs) inoculated with mixed cultures for wastewater treatment. Carbon cloth (CC) anodes were modified by electrochemical oxidation in three electrolytes: nitric acid+sulfuric acid (CC-NS), ammonium nitrate (CC-AN), and ammonium sulfate (CC-AS). The acidic modification of the anode material increased in the ratio of saturated/unsaturated carbon on the surface and consequently, a decrease in electrode resistance was observed. A linear dependence between the MFCs operational characteristics and the anodes resistance (R2 ≥ 0.9) indicated the dominating role of this parameter. This modification also enhanced the bacterial attachment (wet and dry biomass) and biofilm formation. CC-NS, CC-AS and CC-AN anodes accelerated the start up period of the MFCs and demonstrated higher current and power compared to the unmodified CC. The differences in MFCs electrochemical behavior tended to decrease with time. Principal Components Analysis (PCA) was used to identify the parameters having major influence on the system performance, and the results underlined the positive effect of the surface modification on the MFCs output due to increases in the amounts of unsaturated and oxidized carbon, Electrochemical Accessible Surface Area (ECSA) and bacterial attachment.