Microbial fuel cells implemented in constructed wetlands (CW-MFCs), albeit a relatively new technology still under study, have shown to improve treatment efficiency of urban wastewater. So far the vast majority of CW-MFC systems investigated were designed as lab-scale systems working under rather unrealistic hydraulic conditions using synthetic wastewater. The main objective of this work was to quantify CW-MFCs performance operated under different conditions in a more realistic setup using meso-scale systems with horizontal flow fed with real urban wastewater. Operational conditions tested were organic loading rate (4.9 ± 1.6, 6.7 ± 1.4 and 13.6 ± 3.2 g COD/m2·day) and hydraulic regime (continuous vs. intermittent feeding) as well as different electrical connections: CW control (conventional CW without electrodes), open-circuit CW-MFC (external circuit between anode and cathode not connected) and closed-circuit CW-MFC (external circuit connected). Eight horizontal subsurface flow CWs were operated for about four months. Each wetland consisted of a PVC reservoir of 0.193 m2 filled with 4/8 mm granitic riverine gravel (wetted depth 25 cm). All wetlands had intermediate sampling points for gravel and interstitial liquid sampling. The CW-MFCs were designed as three MFCs incorporated one after the other along the flow path of the CWs. Anodes consisted of gravel with an incorporated current collector (stainless steel mesh) and the cathode consisted of a graphite felt layer. Electrodes of closed-circuit CW-MFC systems were connected externally over a 220 O resistance. Results showed no significant differences between tested organic loading rates, hydraulic regimes or electrical connections, however, on average, systems operated in closed-circuit CW-MFC mode under continuous flow outperformed the other experimental conditions. Closed-circuit CW-MFC compared to conventional CW control systems showed around 5% and 22% higher COD and ammonium removal, respectively. Correspondingly, overall bacteria activity, as measured by the fluorescein diacetate technique, was higher (4% to 34%) in closed-circuit systems when compared to CW control systems.