Realizing the enrichment of functional bacteria in microbial fuel cells (MFCs) for wastewater treatment holds substantial research significance. This study explored a novel method of repeatedly oxygen-rich anode environment to enrich electrotrophic/heterotrophic bacteria (EHB) and denitrifying polyphosphate-accumulating organisms (DPAOs) in membrane-less single-chamber air-cathode (AC) MFCs to treat household wastewater. Repeated accumulation of higher dissolved oxygen (DO) was conducive to enhancing the growth of EHB and DPAOs. The systems achieved the maximum removal of 99% of ammonium, 78% of total inorganic nitrogen and 55% of total phosphorus. Repeated oxygen-rich conditions favored the selection of nitrogen-oxidizing bacteria on both electrodes, such as unclassified_f_Xanthomonadaceae, unclassified_p_Bacteroidota, Nitrosomonas and Nitrospira, thereby increasing nitrate availability for DPAOs like Candidatus Contendobacter, unclassified_c_Actinomycetia as well as other denitrifiers such as Anaerolineales, unclassified_p_Chloroflexi, unclassified_o_Rhodospirillales. The genes nxrAB, narGH and nasC, associated with nitrification and denitrification, and the genes gcd, phoD, ugpQ, glpQ, involved in phosphate metabolism, were up-regulated in presence of repeated DO accumulation, thereby enhancing pollutants removal. This study presents a novel approach for the synchronous removal of nitrogen and phosphorus from domestic wastewater through the enrichment of functional bacteria in the repeatedly oxygen-rich ACMFCs.