Eukaryotic communities play critical roles in the diversity and functions of aquatic ecosystems. However, the fate of norfloxacin (NOR) and its impact on eukaryotic community succession in epiphytic biofilm remain unclear. This study investigated the effects of NOR on the aquatic environment, eukaryotic diversity, community assembly and microbial interactions in microcosmic wetlands dominated by Hydrilla verticillata. About 95 % of NOR was removed but exerted serious negative impacts on ecological environment. High concentrations of antibiotics decreased eukaryotic community richness but increased diversity. The assembly process of the eukaryotic community was shaped by stochastic processes, primarily involving dispersal limitation, whereas more positive co-occurrence correlations were triggered in microbial networks (from 57.1 % to 64.6 %), reducing community stability. In addition, the shift in keystones from Bacillariophyta to Metazoa with increased antibiotic concentrations, accompanied by a functional transformation. The partial least squares path modeling showed antibiotics positively affected nutrients (0.693) and network (0.5), but negatively affected β-diversity (-0.288). These data highlight that the ecological impacts of NOR on phylogenetic turnover in eukaryotes and offer references for promoting phytoremediation.