To understand the effects of acclimation schemes on the formation of anode biofilms, different electrical performances are characterized in this study, with the roles of suspended and attached bacteria in single-chamber microbial fuel cells (MFCs). The results show that the generation of current in single-chamber MFCs is significantly affected by the development of a biofilm matrix on the anode surface containing abundant immobilized microorganisms. The long-term operation with suspended microorganisms was demonstrated to form a dense biofilm matrix that was able to reduce the activation loss in MFCs. Also, a Pt-coated anode was not favorable for the initial or long-term bacterial attachment due to its high hydrophobicity (contact angle = $124^{circ}$), which promotes easy detachment of the biofilm from the anode surface. Maximum power ($655.0;mW/m^2$) was obtained at a current density of $3,358.8;mA/m^2$ in the MFCs with longer acclimation periods. It was found that a dense biofilm was able to enhance the charge transfer rates due to the complex development of a biofilm matrix anchoring the electrochemically active microorganisms together on the anode surface. Among the major components of the extracellular polymeric substance, carbohydrates ($85.7;mg/m^2_{anode}$) and proteins ($81.0;mg/m^2_{anode}$) in the dense anode biofilm accounted for 17 and 19%, respectively, which are greater than those in the sparse anode biofilm.