The acclimation of a nitrifying biofilter is a crucial and time-consuming task for setting up a recirculating aquaculture system (RAS). Gaining a better understanding of the dynamics of the microbial community during the acclimation period in the system could be useful for the development of mature nitrifying biofilters. In this study, high-throughput DNA sequencing was applied to monitor the microbial communities on a biofilter during the acclimation period (7 weeks) in high (100 mg N/L) and low (5 mg N/L) total ammonia nitrogen (TAN) treatments. Both treatments were successful for developing a mature nitrifying biofilter, dominated by Proteobacteria, Bacteroidetes, and Nitrospirae. Complete nitrification was found after 7 days of biofilter acclimation as indicated by decreasing TAN concentration, increasing nitrate concentration, and high abundances of the nitrifying bacteria, Nitrosomonadaceae and Nitrospiraceae. The beta diversity analysis of microbial communities showed different clustering of the samples between high and low TAN treatment groups. A greater abundance of nitrifying bacteria was found in the high TAN treatments (27–51%) than in the low TAN treatment (15–29%). The bacterial diversity in biofilters acclimated at high TAN concentration (Shannon’s index 5.40–6.15) were lower than those found at low TAN treatment levels (Shannon’s index 6.40–7.01). The higher diversity in biofilters acclimated at low TAN concentrations, consisting of Planctomycetes and Archaea, might benefit the nutrient recycling in the system. Although nitrification activity was observed from the first week of the acclimation period, the acclimation period should be taken as at least 6 weeks for full development of nitrifying biofilm. Moreover, the reduction of potentially pathogenic Vibrio on biofilters was found at that period.