The microorganisms in biofloc play vital roles in ensuring the stable operation of a culture system as a basis for mass conversion and energy flow. The characteristics and associated functions of a microbial community are largely influenced by the type of nutrition provided by the biofloc. The heterotrophic and autotrophic approaches are the two main methods that have been developed for culturing biofloc. However, the former method usually requires large amounts of organic matter, while the latter requires a long period of cultivation to achieve the function of the biofloc. Recently, the “hete-auto” method has been proposed, which involves the cultivation of biofloc using a combination of the heterotrophic and the autotrophic methods. This method avoids the negative effects of the above-mentioned methods when used alone. To date, most studies on this subject have focused on changes in water quality and nitrogen budgets, but the characteristics of the microbial community of such biofloc, compared to that obtained with conventional methods, remains unclear. Specifically, the effect of water quality on the microbial community of the “hete-auto” biofloc has not been reported. To fill this research gap, we constructed three Litopenaeus vannamei culture systems using “hete-auto” biofloc (AUTO), heterotrophic biofloc (HETE), and the flow-through method (CON), respectively. In the HETE group, sugar (i.e., an external carbon source) was supplied throughout the experiment; whereas in the AUTO group, sugar was added at the beginning of the experiment until the water quality had stabilized (i.e., ammonia nitrogen). In CON, no carbon source was added. Microbial characteristics of biofloc in the three culture systems were analyzed using high-throughput sequencing technology (Illumina MiSeq, Pudong, Shanghai), and the interactions between the microbial community and aquaculture water environment were also investigated. The results showed that Proteobacteria (24.2%–70.45%) and Bacteroldota (8.45%–28.09%) dominated in all treatments. At the genus level, no significant differences were observed between the groups in the relative abundance of the filamentous bacterium Leucothrix (essential for biofloc construction) (3.60%–7.29%). Notably, 94 OTUs classified as Nitrospirota (i.e., nitrifying bacteria) only existed in the AUTO biofloc; furthermore, the relative abundance of nitrifying genes such as AmoA and AmoB in the AUTO biofloc (i.e., 0.17% and 0.20%, respectively) were significantly higher than in the HETE biofloc (i.e., 0.10% and 0.09%, respectively). There were no significant differences in the specific growth rates of L. vannamei cultured with different nutrient types of biofloc. Differences in the microbial composition of the biofloc altered the pathways of nitrogen cycling, resulting in different concentrations of ammonia nitrogen, nitrite, and nitrate, which in turn were influenced by differences in water quality. This study demonstrates that changing the nutrient type results in biofloc with a more rational composition and function, which can effectively control water quality and keep the whole culture system balanced and benign.