Worldwide, the requirement of electrical energy has increased with an increase in population. Thus, there is a need to develop an alternative source of sustainable energy, such as microbial fuel cell (MFC). MFC is a better option of energy generation and can provide a renewable resource which utilizes wastewater into power by the help of microorganisms. MFC is one of the advanced methods for treating wastewater and simultaneously producing current and voltage. Dual-chambered MFC was prepared using two plastic boxes (500 ml) by using wastewater as an anolyte. Different types of mediators are used in MFC including methylene blue, potassium ferricyanide, and EDTA to facilitate and higher the efficiency of electron transfer from the MFC to the electrode. Maximum OCV and current output of sample 1 (Budha Talab pond water) were 0.86 V and 75.1 mA and of sample 2 (Jaypee cement plant) were 1.42 V and 122 mA. The maximum current output of sample 3 (sugar industry, sewage waste, NIT canteen) was 1.3 V. Various physiochemical parameters such as dissolved oxygen (DO), biochemical oxygen demand (BOD), and chemical oxygen demand (COD) were analyzed which affect the power output. The obtained result concluded that wastewater should be feed at a certain time interval to avoid the loss of substrate for organisms in the anodic chamber which lead to the death of the microorganism. Among all, sugar industry wastewater has a high potential for power generation as their physiochemical results are suitable for better power output.