Biological filtration plays an important role in a sustainable recirculating aquaculture system. To provide the appropriate conditions for biofilter acclimation, the effects of the salinity and acclimation period on nitrogen removal and microbial diversity were evaluated. Nitrification rate (NR) of the shrimp tank biofilter increased during the first 4 weeks and decreased thereafter. The highest NR was found in the 5 PSU salinity tank at week 2, whereas in the other salinities (15 and 25 PSU) the highest NR was observed at week 4. Meanwhile, the denitrification rate (DNR) increased significantly on week 8 and the highest DNRs were obtained after switching from an aerobic to an anoxic condition with methanol supplement. Under this anoxic condition the biofilter acclimated in seawater had the highest DNR. Changes in the microbial community during immobilization were monitored using Illumina MiSeq DNA sequencing. Proteobacteria and Bacteriodetes were the most abundant phyla found in this study. For nitrifiers, Nitrosomonadaceae were found from the first week of acclimation, while only one operational taxonomic unit (OTU) of Nitrospiraceae was observed. Under the anoxic condition at the end of the shrimp culture, two OTUs of Nitrospiraceae were found in both the 15 and 25 PSU tanks.