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The use of artificial substrates and biofloc technology can favor fish culture and improve water quality. The aim of this study was to evaluate whether artificial substrates and carbon source additions modify the microbial activity of water bodies. The diversity and structure of microflora in the water after adding artificial substrates and carbon sources to the ponds were analyzed using high-throughput sequencing based on the V3-V4 region of 16S rRNA genes. The results showed that there was no difference in the richness and diversity of intestinal microflora between the control and experimental groups. Principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) showed that artificial substrate and carbon source addition changed the structure of the microflora. The results of a linear discriminant analysis (LDA) effect size (LefSe) indicated 11 biomarkers in the EGˍst group. Spearman correlation heatmap analysis showed that environmental factors affected the bacterial communities, and the results of a redundancy analysis indicated that chemical oxygen demand was a critical factor in controlling the bacterial communities in the water. These results provide an understanding of the effect of artificial substrate and carbon source addition on bacterial diversity and community composition in water. [ABSTRACT FROM AUTHOR]