Nitrogen (N) removal from wastewater is essential, but it a process that demands a substantial amount of energy. Therefore, there is an urgent need to develop treatment processes that can conserve and use energy effectively. This study investigated the potential of a single chamber bio-electrochemical system (BES) for ammonium (NH4+) removal. Various NH4+:NO2− ratios (1:1, 1:0.5, and 1:0) were tested at an applied potential of 0.4 V vs. Ag/AgCl. Potential in the reactors (R-1, R-2, and R-3) significantly improved NH4+ removal efficiencies. Specifically, R-1, R-2, and R-3 exhibited removal efficiencies of 68.12%, 64.22%, and 57.86%, respectively. NH4+ oxidation in R-3 involved using a carbon brush electrode as an electron acceptor. Significant electric charge generation was observed in all reactors (R-1, R-2, and R-3) during NH4+ removal. Particularly, the use of a carbon brush as an electron acceptor in R-3 resulted in higher electric charge generation compared to those in R-1 and R-2, where NO2− served as an electron acceptor. Upon NH4+ removal and concurrent electric charge generation, nitrate (NO3−) accumulation was observed in reactors with applied potential (R-1, R-2, and R-3), demonstrating greater accumulation compared to reactors without potential (R-7, R-8, and R-9). The mechanism involves ammonium oxidizing bacteria (AOB) oxidizing NH4+ to NO2−, which is then further oxidized by nitrite-oxidizing bacteria (NOB) to NO3−. ANAMMOX bacteria could directly produce N2 from NH4+ and NO2− or NH4+ could be oxidized to N2 through extracellular electron transfer (EET). A carbon brush electron acceptor reduces NO2− requirement by 1.65 g while enhancing NH4+ oxidation efficiency. This study demonstrates the potential of mixed culture ANAMMOX granules for efficient NO2-free NH4+ removal.