Giant freshwater prawn Macrobrachium rosenbergii is an important aquaculture species in inland areas of East and South China. In the past decade, prawn disease has occurred frequently during the grow-out stage, mainly due to pathogenic bacteria, which have caused different diseases in different regions. Gaoyou City in Jiangsu Province is the core aquaculture zone of M. rosenbergii in China, however the prawns cultivated in this region suffer from “Dixing disease” during summer. This disease causes prawns to die every day, with lower daily mortality over a long period. Since 2012, another disease has appeared, which causes prawns to grow slowly and develop precocity (commonly known as “iron prawn syndrome” prawn). Both these diseases have caused great economic losses to the prawn industry. In the mid-late grow-out stages, Cyanobacteria blooms have caused the pond water to deteriorate, indirectly affecting prawn production. Microbes in the aquaculture environment are closely related to the health of farmed animals, and the flora structure in the intestinal tract and pond environment of healthy prawn are significantly different from those of a diseased prawn pond. To better understand the composition of dominant bacteria in the aquaculture system of M. rosenbergii and improve the management technology, the composition of cultivable bacteria in the prawn and pond water was investigated in Gaoyou City in July, August, and October 2019. Luria-Bertani medium was used to separate and purify the cultivable bacteria from the hepatopancreas, intestines, gills, and pond water of prawn. Subsequently, the isolated bacteria were identified using 16S rRNA gene sequencing. High-throughput sequencing technology was employed to analyze the microbial community composition in a pond of the prawn affected by “iron prawn syndrome ”and in a pond with a bloom of Microcystis aeruginosa. Additionally, the species of antagonistic bacteria, and their effects on prawn pathogens in the aquaculture system, were screened and analyzed using Aeromonas dhakensis as a test pathogen. A total of 605 cultivable bacteria were isolated and 601 strains were successfully identified, belonging to 37 genera and 119 species. Among them, 76 species in 23 genera were isolated from prawn and 81 species in 28 genera were isolated from pond waters. Investigation results confirmed the existence of potential pathogens in the pond system, belonging to genera such as Aeromonas spp., Enterobacter spp., Lactococcus spp., and Vibrio spp. The species composition and proportion of dominant environmental microorganisms in ponds with “iron shell” prawn are similar to those of healthy prawn ponds, but bacteria in the genera Exiguobacterium and Acinetobacter had higher proportions in disease-affected ponds than those in healthy pond . The blooming of M. aeruginosa could affect the species and structure of environmental microflora and lead to an increase in the proportion of Aeromonas bacteria. Four Bacillus spp., namely B. subtilis, B. methylotrophicus, B. velezensis, and B. amyloliquefaciens, were screened as potential probiotics and were found to have a strong antagonistic effect on Aeromonas spp. These results aid in clarifying the composition of bacterial pathogens in the culture system of M. rosenbergii and provide a theoretical basis for establishing prevention and control technologies for prawn disease.