Conductive materials have been incorporated in anaerobic bioreactors to stimulate direct interspecies electron transfer (DIET), a recently discovered microbial electron-sharing network. Previous studies emphasized the significance of electrocatalytic features associated with DIET, while research gaps are remaining in the understanding of how different operating temperatures and substrate variation will affect those electrochemical characteristics. Consequently, this study examined various electrochemical features, including biomass conductivity, pilA gene, heme-binding proteins, and electron transfers rates (kapp) for methanogenic biomass from conductive carbon fibers amended anaerobic bioreactors operated under different temperatures (20, 37, and 55 °C) with substrate variation (ethanol/propionate). Our results demonstrated a distinct temperature-dependent correlation between biomass conductivities, pilA gene, and heme-binding proteins, suggesting operating temperature would influence the relative contributions of various DIET routes (via pili, redox proteins, and conductive materials). When fed with ethanol, the estimated kapp was the highest at 37 °C (0.0033 s−1 vs 1.1 d−1), followed by 20 °C (0.0018 s−1 vs. 0.74 d−1), and 55 °C (0.0012 s−1 vs. 0.29 d−1), which were positively correlated with the estimated methanogenesis rates. While methanogenesis performance was independent of electron transfer rates (0.0026–0.0032 s−1) during operation with propionate; only carbon fibers amended bioreactor operated at 37 °C showed stable performance in response to substrate variation. Thus, it appeared that bioreactors operated under different temperatures might face a different level of instability caused by substrate variation. Overall, our results linked electrocatalytic features of biomass with temperature and substrate variation, suggesting the importance of temperature-dependent optimization of DIET for improving anaerobic digestion of complex organic feedstock.