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ABSTRACT The freshwater pearl mussel (genus Margaritifera) has shown severe declines, while the mussels play important roles in the translocation of nutrients and materials in river water ecosystems. We hypothesized that the biofilm bacterial composition and nutrient flow may reflect the differences in the existence of mussels. We analyzed water from 14 rivers from in multiple regions of Japan, including eight rivers, where the two species of freshwater pearl mussels (Margaritifera laevis and Margaritifera togakushiensis) are predominantly found, to analyze the microbial and nutritional nature of the biofilm artificially formed in the river. Field-produced biofilms, including the bacterial community structure, were examined, using next-generation sequencing of bacterial 16S rRNA gene amplicons followed by analyzing the genomic DNA extracted from the samples, inorganic nitrogen compounds, and chlorophyll a concentration. Compared to those in the control river without freshwater pearl mussels, biofilms of the existing river contained less inorganic nitrogen (ammonia and nitrate), suggesting the involvement of mussels in regulating the river water nutrient flow. Distinct changes were found in biofilms, depending on mussel existence, particularly in biofilms containing fewer photosynthetic bacterial groups, such as Betaproteobacteria and Cyanobacteria. Conversely, bacteria belonging to Bacteroidales in Bacteroidetes and Clostridiales in Firmicutes were predominantly found in biofilm samples where the mussels existed. Mussels alleviated strict nitrogen limitation in streams and possibly caused a concomitant change in the bacterial communities, where populations of bacterial groups exchanging inorganic nitrogen were low. We demonstrate the profound influence of freshwater mussel species on ecosystem processes and community dynamics across rivers. IMPORTANCE The abundance of freshwater unioid mussels exhibited more diverse patterns of inorganic nitrogen flow and bacterial communities than the areas without mussels. This study demonstrates the effect of mussels on different freshwater ecosystem processes with variable organismal densities and biogeochemical factors. Freshwater unionid mussels significantly affect the ecosystem and community dynamics by modulating the relationships, altering nutrient availability, and indirectly manipulating the downstream ecological members, eventually expanding their role in the river ecosystems.