A continuous flow bioreactor was operated for 300 days to investigate partial nitritation (PN) of mature landfill leachate, establishing the long-term performance of the system in terms of the microbial community composition, evolution, and interactions. The stable operation phase (31–300 d) began after a 30 days of start-up period, reaching an average nitrite accumulation ratio (NAR) of 94.43% and a ratio of nitrite nitrogen to ammonia nitrogen (NO2−-N/NH4+-N) of 1.16. Some fulvic-like and humic-like compounds and proteins were effectively degraded in anaerobic and anoxic tanks, which was consistent with the corresponding abundance of methanogens and syntrophic bacteria in the anaerobic tank, and organic matter degrading bacteria in the anoxic tank. The ammonia-oxidizing bacteria (AOB) Nitrosomonas was found to be the key functional bacteria, exhibiting an increase in abundance from 0.27% to 6.38%, due to its collaborative interactions with organic matter degrading bacteria. In-situ inhibition of nitrite-oxidizing bacteria (NOB) was achieved using a combination of free ammonia (FA) and free nitrous acid (FNA), low dissolved oxygen (DO) with fewer bioavailable organics conditions were employed to maintain stable PN and a specific ratio of NO2−-N/NH4+-N, without an adverse impact on AOB. The synergistic relationships between AOB and both denitrifying bacteria and organic matter degrading bacteria, were found to contribute to the enhanced PN performance and microbial community structure stability. These findings provide a theoretical guidance for the effective application of PN-Anammox for mature landfill leachate treatment.