Mixed culture electrochemically active biofilms (EABs) were developed on carbon paper using a sludge with mixed culture bacteria for microscopic and spectroelectrochemical studies because a naturally mixed culture bacterial strain is more applicable than a pure culture strain. EAB development was confirmed by microbial fuel cells (MFCs) by obtaining a constant increase in potential (0.36 V). Microscopic and spectroscopic studies showed that a mixed culture EABs formed on the support. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS), which are nondestructive voltammetry techniques, indicated that the EABs could be source of electrons and used effectively for various purposes. Routine in vivo analysis of electron transfer between bacterial cells and the electrode was performed, providing insight into the extracellular electron transfer (EET) to the electrode. At low scan rates, CV revealed the catalytic electron transfer ability of EAB between the cells and electrode and showed exceptional redox activities in the presence of acetate. DPV and EIS studies showed that EAB in the presence of acetate can charge the surface by producing and storing sufficient electrons, behave as a capacitor, and have features consistent with EET. Finally, microscopic and spectroelectrochemical studies confirmed the development of a mixed culture EAB and the EET kinetics of EABs. These studies suggest that mixed culture EABs can be used effectively as a biogenic reducing tool for various applications.