Free ammonia (FA) plays a significant role in the stable, long-term, completely autotrophic nitrogen removal over nitrite (CANON) system operation. The influence of FA on the CANON process in a sequencing batch biofilm reactor was explored. Under controlled FA concentrations of 5.0 mg L-1 to 10.0 mg L-1, nitrite-oxidizing bacteria (NOB) was inhibited and achieved partial nitrification, which was important for a successful and quick start-up of the CANON process from activated sludge. However, NOB was acclimated to the condition after the process start-up. Ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing bacteria (AnAOB) activities were unaffected when FA concentration was increased from 10 mg L-1 to 17 mg L-1, but NOB was completely inhibited only for a short time. The AOB and AnAOB activities were inhibited and the CANON system was deteriorated when FA concentration reached 30 mg L-1 to 32.5 mg L-1 at pH 8.5, whereas NOB activity was unaffected. Correlation analysis showed that FA concentration higher than 20 mg L-1 resulted in the deterioration of the system.