Short-term initial anolyte pH adjustment can relieve the performance deterioration of the single-chamber air-cathode buffer-free microbial fuel cell (BFMFC) caused by anolyte acidification. Adjusting the initial anolyte pH to 9 in 5 running cycles is the optimum strategy. The relative abundance of the electrochemically active Geobacter in the KCl-pH9-MFC anode biofilm increased from 59.01% to 75.13% after the short-term adjustment. The maximum power density (Pmax) of the KCl-pH9-MFC was elevated from 316.4mW·m−2 to 511.6mW·m−2, which was comparable with that of the PBS-MFC. And, after the short-term adjusting, new equilibrium between the anolyte pH and the anode biofilm electrochemical activity has been established in the BFMFC, which ensured the sustainability of the improved bioelectricity generation performance.