This study was attempted to evaluate the change of microbial community in inoculums, lag, and stationary phase using the community level physiological profiles (CLPP) base on C-substrate utilization. It was to ascertain the characterizing microbial community over time in the enrichment step of microbial fuel cells. Microbial fuel cell is a device that converts chemical energy to electricity with aid of the catalytic reaction of microorganisms using C-substrate included wastewater. Microbial fuel cells enriched by a mixture of anaerobic digestive sludge of the sewage treatment plant and livestock wastewater were used. The current after enrichment was generated about 0.84 ± 0.06 mA. Microbial community in inoculums, lag and stationary phase used amine group, phosphorylated chemical group, and carboxylic acid group (some exclusion). However, phenolic compound did not use by microorganisms in lag and stationary phase. It means that there are not the microorganisms capable of decompose the phenol in microbial fuel cell enriched by livestock wastewater. In case of substrates of amino acid and carbohydrates group, these C-substrates were only used by microorganisms in the stationary phase. It may be that electrochemically active microorganisms (EAM) which we want to know should utilize the better these C-substrates than that of lag phase. This study showed that the electrochemically active bacteria that can be distinguished by electron changes of C-substrate utilization over time could be separated.