While energy consumption is increasing worldwide due to population growth, the fossil fuels are unstable and exhaustible resources for establishing sustainable life. Using biodegradable compounds present in the wastewater produced in industrial process as a renewable source is an enchanting approach followed by scientists for maintaining a sustainable energy production to vanquish this problem for ulterior generations. In this research, bioelectricity generation with whey degradation was investigated in a two-chamber microbial fuel cell with humic acid as anodic electron mediator and a cathode compartment including combinational electron acceptor. Escherichia coli was able to use the carbohydrate originated from whey to generate bioelectricity. The open-circuit potential in absence of mediator was 751.5 mV at room temperature. The voltage was stable for more than 24 h. Humic acid was used as a suitable mediator. In addition, some mixed chemicals were employed as catholyte. Based on polarization curve, the power and current values in the presence of a mixed solution of potassium iodide (KI), ferric chloride [FeCl3 (Ш)] and manganese chloride tetrahydride (MnCl2·4H2O) with doubling of oxidant (oxygen) concentration using agitation with magnet stirrer in cathode compartment without any buffer solution were boosted to 562.9 μW and 1906.1 μA, respectively, and demonstrated the best result for power generation.