Biofilms can be used to improve the water quality in aquaculture ponds, and elucidating the process of microbial succession in biofilms would allow the characterization of metabolic processes and permit optimization. In the present study, microbial succession of a biofilm growing on artificial substrata in a subtropical freshwater pond was investigated by high-throughput sequencing. Providing artificial substrata effectively reduced the concentrations of total nitrogen and total phosphorus in the pond. Relatively stable microbiota were formed after approximately 1 week. The dominant phyla in the mature biofilm were Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. The relative abundances of denitrifiers and phosphorus-removing bacteria, such as those in Comamonadaceae and Neisseriaceae, were significantly increased. The use of avermectin B1 changed the community structure of the microbiota; the microbiota were more similar to those at Week 0 than to those at Week 3. However, the microbial community structure recovered after approximately 1 week. Our results indicate that using artificial substrata can create a habitat for denitrifiers and phosphorus-removing bacteria, and thereby improve pond water quality. This study provided insight into how the use of artificial substrata could improve water quality and elucidated the environment-biofilm relationship in a subtropical freshwater pond.