In this study, a robust partial denitrification/anammox (PD/A) biofilm system was efficiently established within merely 83 days using solely conventional activated sludge as the inoculum, employing a multivariate regulation strategy involving the addition of biofillers, optimization of municipal wastewater feeding ratios, prolongation of hydraulic retention time, and introduction of hydrazine. The performance variability of PD/A system within a long-term, intricate mainstream substrate environment was further investigated by incrementally elevating municipal wastewater proportion (50 % → 75 % → 100 %). The results demonstrated that PD/A system ultimately achieved an impressive total nitrogen removal efficiency of 93.6 % and an effluent total nitrogen concentration of 8.6 mg/L, accompanied by anammox contributing over 85 % to nitrogen removal. The biofilm’s layered spatial structure nurtured Candidatus Brocadia, enabling it to reach an abundance of 2.5 % under high real municipal wastewater loading. Metabolic function analyses revealed that biofilm enhanced the abundance of genes associated with interspecies electron transfer and quorum sensing communication in PD/A consortia after prolonged exposure to municipal wastewater. These findings underscore the significances of interspecific interactions, bacterial communications, and synergistic metabolic traits within PD/A biofilm systems in the stabilization of real municipal wastewater treatment.