Biofloc technology, extensively used in intensive aquaculture systems, can prompt the formation of microbial aggregates. Microplastics (MPs) are detected abundantly in aquaculture waters. This study explored the effects of MPs on biofloc formation, microbial community composition and nitrogen transformation function in simulated biofloc aquaculture production systems. The formation process and settling performance of bioflocs were examined. High-throughput sequencing of 16S and 18S rRNA genes was used to investigate the microbial community compositions of bioflocs. Nitrogen dynamics were monitored and further explained from functional genes and microorganisms related to nitrogen transformation by metagenome sequencing. We found that the aggregates consisting of bioflocs and MPs were formed and the systems with MPs had relatively weak settling performance. No significant differences in bacterial diversity (p > 0.05) but significant differences in eukaryotic diversity (p < 0.05) were found between systems without and with MPs. Significant separations in the microbial communities of prokaryotes (p = 0.01) and eukaryotes (p = 0.01) between systems without and with MPs were observed. The peak concentration of nitrite nitrogen (NO2−-N) in systems with MPs was lower than that in systems without MPs (pControl/MPs Low = 0.02 and pControl/MPs High = 0.03), probably due to the low abundance of hao and affiliated Alphaproteobacteria_bacterium_HGW-Alphaproteobacteria-1 and Alphaproteobacteria_bacterium, but the high abundance of nxrA and affiliated Alphaproteobacteria_bacterium_SYSU_XM001 and Hydrogenophaga_pseudoflava that related to nitrification. The low concentration of NO2−-N in systems with MPs suggested that the presence of MPs might inhibit ammonia oxidation but promote nitrite oxidation by altering the microbial community structure and function. These results indicated that aggregates consisting of bioflocs and MPs could be formed in aquaculture water, and thus, inhibiting their settlement and altering nitrogen transformation function by affecting the microbial community composition.