Bacteria capable of complete ammonia oxidation (comammox) are widespread and contribute to nitrification in wastewater treatment facilities. However, their roles in partial nitrification-anaerobic ammonium oxidation (anammox) systems remain unclear. In this study, a bench-scale bioreactor with continuous stirring was operated for more than 1000 days with limited oxygen supply to achieve efficient nitrogen removal (70.1 ± 2.7%) at a low ammonium loading of 35.2 mg-N/L/day. High-throughput amplicon sequencing analysis of the comammox ammonia monooxygenase subunit A (amoA) gene revealed seven sequence types from two clusters in clade A of comammox Nitrospira. Quantitative polymerase chain reaction analyses suggested that the comammox species dominated the ammonia-oxidizing community, with an abundance as high as 89.2 ± 7.9% in total prokaryotic amoA copies. Multiple linear regression further revealed the substantial contribution of the comammox Nitrospira to ammonia oxidation in the bioreactor. The investigation with bioreactor and batch experiments consistently showed that activities of comammox Nitrospira were inhibited by free ammonia far more severely than other ammonia-oxidizing microbes. Overall, this study provided new insight into the ecology of comammox Nitrospira under hypoxic conditions and suggested comammox-associated partial nitrification-anammox as a potential method for treating low-strength ammonium-containing wastewater.