Chlorella pyrenoidosa is a widely used microalgae in the aquaculture process of Litopenaeus vannamei. In order to evaluate the effects of supplementing C. pyrenoidosa on water quality, shrimp growth performance, and biofilm bacterial community structure in L. vannamei aquaculture systems, C. pyrenoidosa suspension was supplemented in the precultured biofilm (PB) and water exchange (WE) culture systems. The results showed that supplementation with C. pyrenoidosa significantly increased the turbidity and the concentrations of total ammonia nitrogen (TAN) and nitrite nitrogen (
NO
2
−
─N) in the PBC system (the PB system supplemented with C. pyrenoidosa), but had no significant effect on those in the WEC system (the WE system supplemented with C. pyrenoidosa). However, the TAN concentration of all systems remained at a low level (<0.1 mg/L) throughout the experiment. Additionally, supplementation with C. pyrenoidosa decreased the final concentrations of total phosphorus in the PBC and WEC systems by 15.74% and 23.33%, respectively. With the supplementation of C. pyrenoidosa, the final weight, final biomass, and average daily growth rate of L. vannamei increased, while the feed conversion ratio decreased. 16srRNA high-throughput sequencing results showed that the supplementation of C. pyrenoidosa to the PBC system resulted in a decrease in the relative abundances of Proteobacteria and Bacteroidetes and an increase in the relative abundance of Acidobacteria. Compared to those in the initial biofilm sample, the relative abundances of nitrifying bacteria, Nitrosococcus, Nitrosomonas, Nitrococcus, and Nitrospira, in PBCF (biofilm in the PB systems with C. pyrenoidosa) and PBOF (biofilm in the PB systems without C. pyrenoidosa) decreased from 10.18% to 3.05% and 0.75%, respectively. In conclusion, C. pyrenoidosa, as a supplement to the aquaculture systems, has practical application prospects.