The role of operating conditions, specifically duty cycling, on microbial fuel cell (MFC) performance has only recently been studied. This study uses an environmental MFC containing multiple anodes, positioned in chambered anoxic seawater overlying marine sediments, to explore how varying the time an anode is connected and disconnected from a cathode influences cumulative charge, and finds that a switching interval of 15 seconds when duty cycling among the anodes results in the greatest normalized current density (amps per unit time connected). Further studies demonstrate the overall electrode reaction resistance increases with shorter disconnection times, and there is a minimum time below which current decreases significantly. Microbial diversity analyses reveal a substantial enrichment in sulfur-cycling microbes on the anodes compared to the sediment, though no measurable change in community composition among operational anodes. Based on these data, we posit that, in environmental MFCs, replenishment of depleted chemical species within the biofilm and surrounding diffusion layer is necessary for maximum charge transfer, and that proton flux is not limiting in highly buffered systems. These data underscore the importance of considering all factors, including diffusion and migration flux, microbial community composition, and resting time, when optimizing MFC performance.