Appropriate inoculation and maturation may be crucial for shortening the startup time and maximising power output of Microbial Fuel Cells (MFCs), whilst ensuring stable operation. In this study we explore the relationship between electrochemical parameters of MFCs matured under different external resistance (Beet) values (50 Omega 10 k Omega) using non-synthetic fuel (human urine). Maturing the biofilm under the lower selected R-ext results in improved power performance and lowest internal resistance (R-int), whereas using higher R-ext results in increased ohmic losses and inferior performance. When the optimal load is applied to the MFCs following maturity, dependence of microbial activity on original R-ext values does not change, suggesting an irreversible effect on the biofilm, within the timeframe of the reported experiments. Biofilm microarchitecture is affected by R-ext and plays an important role in MFC efficiency. Presence of water channels, EPS and precipitated salts is distinctive for higher R-ext and open circuit MFCs. Correlation analysis reveals that the biofilm changes most dynamically in the first 5 weeks of operation and that fixed R-ext lefts an electrochemical effect on biofilm performance. Therefore, the initial conditions of the biofilm development can affect its long-term structure, properties and activity.