One of the important factors in enhancing the performance of microbial fuel cells (MFCs) is reactor design and configuration. Therefore, this study was conducted to evaluate the regressors and their operating parameters affecting the double anode chamber–designed dual-chamber microbial fuel cell (DAC-DCMFC) performance. Its primary design consists of two anode chamber compartments equipped with a separator and cathode chamber. The DAC-DCMFCs were parallelly operated over 8 days (60 days after the acclimation period). They were intermittently pump-fed with the different organic loading rates (OLRs), using chemically enriched sucrose as artificial wastewater. The applied OLRs were adjusted at low, medium, and high ranges from 0.4 kg.m−3.d−1 to 2.5 kg.m−3.d−1. The reactor types were type 1 and type 2 with different cathode materials. The pH, temperature, oxidation-reduction potential (ORP), optical density 600 (OD600), chemical oxygen demand (COD), and total organic carbon (TOC) were measured, using standard analytical instruments. In general, the power production achieved a maximum of 866 ± 44 mW/m2, with a volumetric power density of 5.15 ± 0.26 W/m3 and coulombic efficiency of 84%. Two-stage COD and TOC removal at medium OLR achieved a range of 60–80%. Medium OLR is the recommended level to enhance power production and organic removal in DAC-DCMFC. The separated anode chambers into two parts in a dual anode chamber microbial fuel cell adjusted by various organic loadings expressed a preferable comprehension in the integrated MFCs for wastewater treatment.