In recirculating aquaculture systems (RAS), a significant challenge involves managing the biological cycling of nitrogenous waste. The biofloc technique (BFT) represents a recent advancement technology, enabling high-density culture with limited or zero water exchange. However, there is still a lack of information concerning the microbial diversity derived from BFT, particularly in relation to nitrogen cycling. We hypothesized that in a RAS, the nitrification cycle would become fully functional after a distinct adjustment period following BFT treatment, and that the microbiome structure during this period would differ significantly from that during subsequent stabilization periods. To investigate this, we observed the dynamics of freshwater microbiome and water properties subsequent to BFT and ammonium chloride treatment during the pre-treatment maturation phase for Japanese eel cultivation using 16S rRNA gene amplicon sequencing and nitrifier-specific qPCR. Our research demonstrated that water properties and freshwater microbiome underwent dynamic alterations over time. Through random forest analysis, we identified that nitrate, final product of the nitrification cycle contributes most significantly to microbiome diversity. Building on this, we elucidated the relationships between nitrogen compounds, nitrifier abundance, and microbiome diversity in the nitrification cycle using structural equation modeling. Furthermore, we observed a sharp increase in nitrifier abundance starting from three weeks after BFT treatment, and we determined that several microbiome structure indices significantly differ between the adjustment period and stabilization period. Our study provides a detailed and multifaceted understanding of the changes induced by BFT in RAS, highlighting its potential application in promoting water quality through nitrification and subsequent microbiome alterations.