Biofloc technology (BFT) systems heavily rely on microbiota to mitigate ammonia toxicity and manage essential nutrient cycling. Understanding the diversity and functional role of microbiota within BFT-applied aquaculture systems is crucial for ensuring sustainable operations. Though some studies exist on BFT microbiota, research on microbial differences in Japanese eel aquaculture is still limited, hindering the wider application of BFT systems. In this study, we analyzed the characteristics of water quality factors and microbiota in Japanese eel (Anguilla japonica) breeding water, applying the BFT system. Using a metabarcoding approach, the diversity and community structure of aquatic microbiota were compared between BFT and continuous flow (CF) systems. The pH was significantly higher in CF water, while total ammonia nitrogen (TAN) and nitrite (NO2−-N) was higher in BFT water. Alpha diversity was significantly higher in BFT compared to CF systems, and it was correlated significantly with pH and TAN. In both BFT and CF water, the phyla Proteobacteria and Bacteroidota were found to be the most abundant. In the BFT water, a diverse array of bacterial taxa, including BFT-specific clades, were consistently present, while the microbiota in CF water was more variable and contained fewer specific taxa. In addition, bacterial functions related to nitrate reduction, sulfur compound oxidation, and chitinolysis were significantly more abundant in BFT than in CF systems. These findings highlight differences in water quality and microbiota between aquaculture systems, which can inform future research on the use of BFT for sustainable fish farming.