Microbial fuel cells (MFCs) have attracted attention by directly converting the bioelectrochemical energy possessed by the organic materials that make up the biomass into electrical energy. In this study, the relationship between the biofilm formed on the titanium-based anode electrode surface, and the chemical composition of the substrate, the energy source of MFC, was investigated. For this, MFCs were made by using poplar wood shavings rich in organic material as the substrate, titanium-based material as the anode electrode, and natural soil as bacterial habitat. Three types of MFCs containing 1%, 10%, and 20% poplar wood shavings by weight were made and named P1-MFC, P2-MFC, and P3-MFC, respectively. According to electrochemical analysis, P3-MFC provided the highest open circuit voltage with 490 mV value, and the highest power density with 5.11 mW/m2 value compared to other MFCs. According to optical microscopy examinations, there were Bacillus and Coccus species of bacteria in the soil structure, and these bacteria also existed around the fiber of poplar wood shavings in MFCs. Scanning electron microscopy (SEM), energy-dispersive spectrum (EDS), and Fourier transform infrared spectroscopy (FTIR) analysis showed that MFCs formed biofilm in the titanium-based anode, and the chemical composition of this biofilm with poplar tree was similar. As a result, due to the catalysis reactions of bacteria, the titanium-based anode electrode surface was coated with polymer biofilm released from poplar wood shavings.