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The indoor recirculating aquaculture system (RAS) is a sustainable alternative to traditional aquaculture systems, and a biofilter is a crucial component for the stable operation of this system. In this study, the bacterial communities within different sections of the biofilter in a full‐scale RAS for the culture of Tongue Sole Cynoglossus semilaevis were investigated using polymerase chain reaction–based 454 pyrosequencing. A total of 67,368 effective sequences of the 16S rRNA gene with V1–V3 regions were obtained from samples collected from the front section, rear section, and sludge of the biofilter. Using a 3% cut‐off level and sufficient sequencing depth, a total of 5,185 operational taxonomic units (OTUs) were identified in these sequences across all samples. However, a low shared richness of 570 OTUs (11.0% of the reads) was found in all samples, indicating a high diversity of bacterial profiles even in a single biofilter. Based on the SILVA database classifier of phylogenetic taxa, Proteobacteria (average, 35.6%) and Bacteroidetes (average, 30.7%) were found to be the predominant species in all samples, followed by Nitrospirae (6.0%) in the front section, Acidobacteria (7.9%) in the rear section, and Planctomycetes (9.7%) in the sludge. Overall, 404 genera were assigned to all samples. But among the 10 most common genera, only Nitrospira, Owenweeksia, and Marinicella were found in all samples. The results of the phylogenetic analysis suggest that the nitrifying groups of ammonia‐oxidizing bacteria belong to Nitrosomonas clusters, and the nitrite‐oxidizing bacteria are related to Nitrospira and Nitrospina clusters. In summary, our results indicate that the RAS fixed‐film biofilter is a highly species‐rich ecosystem. For this reason, a potential precision‐operated strategy to manage the microbial resources in the biological treatment section in an RAS should be adopted, with consideration being given to the peculiarities and requirements of the species being cultured.

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