Abstract:[Background] The Challenger Deep in the Mariana Trench is the deepest known site in the earth’s ocean. Understanding of its microbial community structure and bacterial isolation are important for mining of deep-sea genetic resources. [Objective] Various types of bacteria were isolated from water samples at different depths in the Mariana Trench, and these microbes were compared with high-throughput sequencing results to know microbes that should be further isolated. [Methods] Different media were used to isolate bacteria from water samples, and Illumina Miseq high-throughput sequencing system was used for the diversity analysis of bacteria and archaea. [Results] A total of 783 strains were isolated from 6 water samples at different depths and stations. These isolates belonged to 4 phyla, 6 classes and 28 genera. Among them, Proteobacteria was dominant and 67.8% of total strains belonged to Gammaproteobacteria. The microbes were mainly belonged to Sulfitobacter, Pseudomonas and Pseudoalteromonas. These types of microbes were widely distributed in all samples and detected by high-throughput sequencing. High-throughput sequencing results showed that Cyanobacteria was the major bacteria in samples at shallow depth and Proteobacteria were dominated in other samples. The microbial community structures of samples at different depths were quite different. [Conclusion] Not only bacteria with relatively high abundance in Mariana Trench water were isolated, but also some bacteria with relatively low abundance. The isolated microbes in the Mariana Trench water samples will be helpful for the mining of functional microbes and enzymes.