Aims To investigate community shifts of amoA-encoding archaea (AEA) and ammonia-oxidizing bacteria (AOB) in biofilter under nitrogen accumulation process. Methods and Results A laboratory-scale rockwool biofilter with an irrigated water circulation system was operated for 436days with ammonia loading rates of 4963 NH3gm3day1. The AEA and AOB communities were investigated by denaturing gradient gel electrophoresis, sequencing and real-time PCR analysis based on amoA genes. The results indicated that changes in abundance and community compositions occurred in a different manner between archaeal and bacterial amoA during the operation. However, both microbial community structures mainly varied when free ammonia (FA) concentrations in circulation water were increasing, which caused a temporal decline in reactor performance. Dominant amoA sequences after this transition were related to Thaumarchaeotal Group I.1b, Nitrosomonas europaea lineages and one subcluster within Nitrosospira sp. cluster 3, for archaea and bacteria, respectively. Conclusions The specific FA in circulation water seems to be the important factor, which relates to the AOB and AEA community shifts in the biofilter besides ammonium and pH. Significance and Impact of the Study One of the key factors for regulating AEA and AOB communities was proposed that is useful for optimizing biofiltration technology.