An upflow anaerobic biofilter (AF) was developed to investigate anaerobic ammonium-oxidizing (ANAMMOX) efficiency in treating low-strength wastewater at ambient temperature (15.3–23.2 °C). Denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization were used to investigate treatment effects on the microbial community. Stepwise decreases in influent ammonia concentration could help ANAMMOX bacteria selectively acclimate to low-ammonia conditions. With an influent ammonia concentration of 46.5 mg/L, the AF reactor obtained an average nitrogen removal rate of 2.26 kg/(m3 day), and a removal efficiency of 75.9%. polymerase chain reaction-DGGE results showed that microbial diversity in the low matrix was greater than in the high matrix. Microbial community structures changed when the influent ammonia concentration decreased. The genus of functional ANAMMOX bacteria was Candidatus Kuenenia stuttgartiensis, which remained stationary across study phases. Visual observation revealed that the relative proportions of ANAMMOX bacteria decreased from 41.6 to 36.3% across three study phases. The AF bioreactor successfully maintained high activity due to the ANAMMOX bacteria adaptation to low temperature and substrate conditions.