Seamounts are widespread underwater topographic features in the ocean that exert an influential role in shaping the microbial biogeographic distribution. Nevertheless, research on the differences in microbial biogeographic distribution between seamount and non-seamount upper water column is still lacking, particularly in the Indian Ocean where studies are limited. In the present study, a total of 45 seawater samples were collected from the water column (5-200 m) of seamounts (HS) and non-seamounts (E87 transect) regions in the Eastern Indian Ocean (EIO) for the analysis of microbial biogeographic patterns and community assembly processes. The results indicated that bacterial community diversity did not differ significantly between the HS and E87 transect regions; however, the community composition was significantly different. Additionally, bacterial community diversity, composition, as well as structure were more affected by depth than by region. Community diversity tended to increase with depth in E87 transect region, while it tended to decrease in HS region. A distance decay analysis also demonstrated that bacterial communities were more influenced by environmental and depth distances than geographic distances. In the assembly of bacterial communities on HS and E87 transect regions, as well as at different depths, stochastic processes, particularly dispersal limitation, were found to be predominant. These findings enhance our comprehension of bacterial community characteristics in the upper seawater of seamounts and non-seamounts regions in the EIO and offer insights into the assembly processes shaping microbial communities at varying depths. IMPORTANCE By comparing the bacterial diversity, composition, and structure in the upper seawater of seamount and non-seamount areas, we provide valuable insights into the influential role of seamounts in shaping microbial biogeography. The finding that the depth had a more significant impact on bacterial community characteristics than region underscores the importance of considering vertical stratification when examining microbial distributions. Moreover, the dominance of stochastic processes, particularly dispersal limitation, in governing community assembly across both seamount and non-seamount areas offers critical implications for the mechanisms underlying microbial biogeographic patterns in these dynamic ocean environments. This study expands the current knowledge and lays the groundwork for further investigations into the complex interactions between oceanographic features, environmental gradients, and microbial community dynamics in the Indian Ocean.