Abstract Microbial fuel cells (MFCs) have applications other than electricity production, including the capacity to power desirable reactions in the cathode chamber. However, current knowledge of the microbial ecology and physiology of biocathodes is minimal, and as a result more research dedicated to understanding the microbial communities active in cathode biofilms is required. Here we characterize the microbiology of denitrifying bacterial communities stimulated by reducing equivalents generated from the anodic oxidation of acetate. We analyzed biofilms isolated from two types of cathodic denitrification systems: (1) a loop format where the effluent from the carbon oxidation step in the anode is subjected to a nitrifying reactor which is fed to the cathode chamber and (2) an alternative non-loop format where anodic and cathodic feed streams are separated. The results of our study indicate the superior performance of the loop reactor in terms of enhanced current production and nitrate removal rates. We hypothesized that phylogenetic or structural features of the microbial communities could explain the increased performance of the loop reactor. We used PhyloChip with 16S rRNA (cDNA) and fluorescent in situ hybridization to characterize the active bacterial communities. Our study results reveal a greater richness, as well as an increased phylogenetic diversity, active in denitrifying biofilms than was previously identified in cathodic systems. Specifically, we identified Proteobacteria, Firmicutes and Chloroflexi members that were dominant in denitrifying cathodes. In addition, our study results indicate that it is the structural component, in terms of bacterial richness and evenness, rather than the phylogenetic affiliation of dominant bacteria, that best corresponds to cathode performance.