Although various membrane and cathode electrode types have been investigated in dual chamber microbial fuel cells (DCMDCs), there is no agreement on which combination of electrodes and separator materials works best. The main objective of this study is to investigate the synergistic effects of cathode electrodes and membranes on the performance of DCMFCs during the start-up phase, to model the DCMFCs through artificial neural networks (ANN), and to determine the most cost-effective choice. Principal component analysis was used to analyze the interrelationships among various factors that influence DCMFCs. Two types of ion exchange membranes (cation exchange membrane and anion exchange membrane) and two types of cathode electrodes (carbon cloth covered with 20% Pt as catalyst and plain carbon cloth) were studied. Electrodes and membranes were analyzed using AFM, TEM, SEM, XRF, and FTIR to investigate changes in morphology and the potential of fouling. The results showed that DCMFC with cation exchange membrane (CEM) and plain carbon cloth (CC) generated the maximum power among the investigated configurations. The average voltage values and COD removal efficiency reached 748 ± 13 mV and 57.8 ± 2.5%, respectively. The maximum power density achieved was obtained when the external resistance was set at 1000 Ω and was 180 mA/m2. ANN was successful in predicting the output voltage for the examined DCMFCs. The use of DCMFC with CC and CEM can be considered optimal in terms of performance and cost.