Prokaryotes play a key role in particulate organic matter's decomposition and remineralization processes in the vertical scale of seawater, and prokaryotes contribute to more than 70% of the estimated remineralization. However, little is known about the microbial community and metabolic activity of the vertical distribution in the trenches. The composition and distribution of prokaryotes in the water columns and benthic boundary layers of the Kermadec Trench and the Diamantina Trench were investigated using high-throughput sequencing and quantitative PCR, together with the Biolog EcoplateTM microplates culture to analyze the microbial metabolic activity. Microbial communities in both trenches were dominated by Nitrososphaera and Halobacteria in archaea, and by Alphaproteobacteria and Gammaproteobacteria in bacteria, and the microbial community structure was significantly different between the water column and the benthic boundary layer. At the surface water, amino acids and polymers were used preferentially; at the benthic boundary layers, amino acids and amines were used preferentially. Cooperative relationships among different microbial groups and their carbon utilization capabilities could help to make better use of various carbon sources along the water depths, reflected by the predominantly positive relationships based on the co-occurrence network analysis. In addition, the distinct microbial metabolic activity detected at 800 m, which was the lower boundary of the twilight zone, had the lowest salinity and might have had higher proportions of refractory carbon sources than the shallower water depths and benthic boundary layers. This study reflected the initial preference of the carbon source by the natural microbes in the vertical scale of different trenches and should be complemented with stable isotopic tracing experiments in future studies to enhance the understanding of the complex carbon utilization pathways along the vertical scale by prokaryotes among different trenches.