A microbial fuel cell (MFC) system containing modular half-submerged biocathode was operated for 6 months in an 800 L flow-through system with domestic wastewater. For the first time, spatial and temporal differences in biofilm communities were examined on large three-dimensional electrodes in a wastewater MFC. Biocathode microbial community analysis showed a specialized biofilm community with electrogenic and electrotrophic taxa forming during operation, suggesting potentially opposing electrode reactions. The anodic community structure shifted during operation, but no spatial differences were observed along the length of the electrode. Power output from the system was most strongly influenced by pH. Higher power densities were associated with the use of solids-dewatering filtrate with increased organic matter, conductivity, and pH. The results show that the biocathode was the rate-limiting step and that future MFC design should consider the effect of size, shape, and orientation of biocathodes on their community assembly and electrotrophic ability.