Microbial electrochemical cell (MXC) technology is a source of sustainable energy which comes from microorganisms. Recent advances in the fields of electromicrobiology and electrochemistry with focus on microbial electrolysis cells (MECs) has earned this technology its name as alternate “green energy”. Despite advances, this technology is still facing challenges to address low power and current density output. Thermoanaerobacter pseudethanolicus 39E (ATCC 33223), a thermophilic, Fe(III)-reducing, and fermentative bacterium, was evaluated for its ability to produce current from four electron donors xylose, glucose, cellobiose, and acetate with a fixed anode potential (+ 0.042 V vs SHE) in a microbial electrochemical cell (MXC). Under thermophilic conditions (60 °C), T. pseudethanolicus produced high current densities from xylose (5.8 ± 2.4 Am−2), glucose (4.3 ± 1.9 A m−2), and cellobiose (5.2 ± 1.6 A m−2). It produced insignificant current when grown with acetate, but consumed the acetate produced from sugar fermentation to produce electrical current. Low-scan cyclic voltammetry (LSCV) revealed a sigmoidal response with a midpoint potential of −0.17 V vs SHE. Coulombic efficiency (CE) varied by electron donor, with xylose at 34.8% ± 0.7%, glucose at 65.3% ± 1.0%, and cellobiose at 27.7% ± 1.5%. Anode respiration was sustained over a pH range of 5.4−8.3, with higher current densities observed at alkaline pH values. Scanning electron microscopy showed a well-developed biofilm of T. pseudethanolicus on the anode, and confocal laser scanning microscopy demonstrated a maximum biofilm thickness (Lf) greater than ~150 μm for the glucose-fed biofilm. Microbial electrochemical cells (MXCs) are devices powered by microorganisms to generate electricity via oxidation of organic substrates. It is critical to understand the significance of sediment inocula in forming anodic biofilms to improve MEC performance. Five environmental samples were evaluated for electrical current production using acetate-fed microbial electrolysis cells (MECs). Three of these samples were able to produce significant current densities ranging between 3 to 6.3 Am-2. 16S rDNA targeted deep sequencing comparisons of anodic biofilms and sediment bacterial community structures revealed significant differences in bacterial community structures. Bacterial community producing the highest current density x after enrichment was dominated by the class Bacteroidia, δ-proteobacteria and Erysipelotrichi. Comparison of phylogenetic information of bacterial communities with 7 previously reported enriched samples by reconstruction of unobserved states (PICRUSt) analysis clearly distinguished the biofilm communities from the sediment inocula in terms of higher abundance of genes related to anode respiration. Principal Coordinate Analysis (PCoA) also indicated that the clustering of biofilm communities was in accordance with the predominant genera in each sample, such as Geobacter dominating one cluster of biofilms. All the sediments formed a single cluster, which included the Carolina mangrove biofilm community which showed only minor changes from its originating sediment community after enrichment. Predominantly, high current densities are associated with the enrichment of a few microorganisms, often within a single family; however, this organism can be different depending on the inoculum source. Because the selective enrichment selects for just a few bacteria, the biofilm community is significantly different from that of the sediment. While δ-proteobacteria (or the family Geobacteraceae) is dominant in many samples producing high current densities, other samples show communities with yet unidentified ARB as the major fraction.