In order to study the correlation between microbial composition of intestinal tract and aquaculture environment of Oreochromis niloticus and streptococcicosis, 16S rDNA high-throughput sequencing method was used to analyze the microflora structure of intestinal tract of healthy and diseased O. niloticus and that of their pond water and sediment. The results showed that the microbial diversity of sediment was higher than that of pond water and O. niloticus intestinal tract. The microbial composition of O. niloticus intestinal tract was more similar to pond water than to sediment. The microbial diversity of O. niloticus intestinal tract and sediment in the disease group was lower than that in the healthy group, while the microbial diversity of pond water in the disease group was higher compared with the healthy group. The OTU cluster analysis revealed that significant difference only existed in intestinal microorganism between the disease group and the health group. The majority of healthy intestinal samples were clustered together. The microflora structure analysis showed that the dominant bacteria of pond water and sediment in the healthy group and the disease group were similar. The abundance of proteobacteria and fusobacteria was far higher in the disease group of pond water, while the abundance of verrucomicrobia and planctomycetes was obviously lower compared with the healthy group of pond water. The abundance of flora with the function of organic degradation and ecologic restoration (such as Anaeromyxobacter, Methanosaeta and Virgibacillus) was far lower in the sediment of disease group, while the abundance of pathogenic bacteria (such as Mannheimia) was higher compared with the sediment of healthy group. The abundance of pathogenic bacteria (such as Streptococcus, Mycobacterium and Mannheimia) in intestinal tract of disease group was higher, while the abundance of probiotics (such as Lactococcus, Cetobacterium and Rhodococcus) was lower compared with intestinal tract of healthy group. Streptococcus agalactiae was detected not only in aquaculture environment and intestinal tract of the disease group but also in those of the healthy group. The abundance of S. agalactiae in intestinal tract of the disease group was significantly higher than that in the healthy group, while there was no significant difference between the disease group and the health group either for pond water or sediment. In conclusion, the variation of the abundance of pathogenic bacteria and probiotics in pond water and sediment revealed that the aquaculture environment of the diseased pond was deteriorated. The outbreak of streptococcicosis was closely related to the rapidly increased abundance of S. agalactiae in the intestinal tract of O. niloticus after its microecological balance had been destroyed. The increased abundance of S. agalactiae in the intestinal tract of infected O. niloticus had no direct link with its abundance in pond water or sediment.