Trimethylamine N-oxide (TMAO) is one of the most important nutrients for bacteria in the deep-sea environment and is capable of improving pressure tolerance of certain bacterial strains. To assess the impact of TMAO on marine microorganisms, especially those dwelling in the deep-sea environment, we analyzed the bacterial community structure of deep-sea sediments after incubated under different conditions. Enrichments at 50 MPa and 0.1 MPa revealed that TMAO imposed a greater influence on bacterial diversity and community composition at atmospheric pressure condition than that under high hydrostatic pressure (HHP). We found that pressure was the primary factor that determines the bacterial community. Meanwhile, in total, 238 bacterial strains were isolated from the enrichments, including 112 strains affiliated to 16 genera of 4 phyla from the Yap Trench and 126 strains affiliated to 11 genera of 2 phyla from the Mariana Trench. Treatment of HHP reduced both abundance and diversity of isolates, while the presence of TMAO mainly affected the diversity of isolates obtained. In addition, certain genera were isolated only when TMAO was supplemented. Taken together, we demonstrated that pressure primarily defines the bacterial community and culturable bacterial isolates. Furthermore, we showed for the first time that TMAO had distinct influences on bacterial community depending on the pressure condition. The results enriched the understanding of the significance of TMAO in bacterial adaptation to the deep-sea environment.