Sludge treatment using free ammonia (FA) is an innovative approach that was recently reported effective achieving stable mainstream nitrogen removal via the nitrite pathway. This study aims to investigate the adaptation of nitrifying community and the response of nitrification performance to high-level of FA exposure under real wastewater conditions. Two parallel lab-scale sequencing batch reactors were operated and fed with real municipal wastewater, with one receiving sludge treatment by FA and another as a control. While the FA approach rapidly achieved partial nitrification with a nitrite accumulation ratio (NAR) of approximately 60%, the partial nitrification eventually failed due to nitrite-oxidizing bacteria (NOB) adaptation to FA inactivation. NOB activity in the inoculum was suppressed by 82% after exposure to FA at ~220 mg NH3-N/L. However, towards the end of the experiments, significantly higher NOB activities were observed after exposure to the same level of FA. Distinct behaviours of NOB observed in batch tests during the study supported the reactor operational data and strongly suggested the adaptation of NOB under the FA stress. Furthermore, microbial community analysis revealed the underlying mechanism of the observed adaptation: the dominant NOB changed from Nitrospira to Candidatus Nitrotoga. It is for the first time shown that Ca. Nitrotoga are highly resistant to FA inhibition and inactivation in comparison to Nitrospira and Nitrobacter. In addition, while the Nitrosomonas genus was always the dominant ammonia-oxidizing bacteria (AOB) throughout the study, different shift in a species level was observed. Display omitted •Inactivation and adaptation of nitrifiers exposed to FA were investigated.•Dominant NOB genus shifted from Nitrospira to Ca. Nitrotoga under FA stress.•Ca. Nitrotoga are resistant to FA inhibition/inactivation compared with other NOB.•Nitrosomonas were always dominant AOB genus under FA stress.