To examine the eukaryotic biodiversity of aquatic ecosystems in the Qiantang River, China, eukaryotic microbes in the river were investigated using 18S rRNA gene sequencing during the breeding season (July to August 2018). Four distinct distribution patterns (1. Jiande; 2. Tonglu and Fuyang; 3. Jiubao; 4. Yanguan) of the microbial community and their potential effects on fishery activities were observed. Results show lower abundances of Dinophyta and Fungi and higher abundances of Cryptophyta and Chlorophyta in Tonglu and Fuyang than those in the other three sections. In addition, the reserves (Tonglu and Fuyang) destabilized the original eukaryotic microbial co-occurrence network. Among all the environmental factors measured, nitrogen (nitrite, nitrate, ammonium), water temperature and total chlorophyll a acted as major driving factors that controlled the eukaryotic microbial distribution. Furthermore, the existence of some algae (e.g., Chrysophyceae, Cryptophytes, and Chlorophyceae) and fungi (e.g., Rhizophydium) in Tonglu and Fuyang was beneficial to juvenile fish growth and water quality, although some detrimental species (e.g., Aphanomyces) needed attention. This study provides further insights into the sustainable protection and utilization of rivers.