In freshwater microbial ecology, extensive studies are attempting to characterize the vast majority of uncultivated bacterioplankton taxa. However, these studies mainly focus on the epilimnion and little is known regarding the bacterioplankton inhabiting the hypolimnion of deep holomictic lakes, despite its biogeochemical importance. In this study, we investigated the bacterioplankton community composition in a deep freshwater lake with a fully oxygenated hypolimnion (Lake Biwa, Japan) using high‐throughput 16S rRNA gene amplicon sequencing. Sampling at a pelagic site over 15 months throughout the water column revealed that the community composition in the hypolimnion was significantly different from that in the epilimnion. The bacterial community in the hypolimnion was composed of groups dominating in the whole water layer (e.g., bacI‐A1 and acI‐B1) and groups that were hypolimnion habitat specialists. Among the hypolimnion specialists, members of Chloroflexi and Planctomycetes were highly represented (e.g., CL500‐11, CL500‐15 and CL500‐37), followed by members of Acidobacteria, Chlorobi and nitrifiers (e.g., Ca. Nitrosoarchaeum, Nitrosospira and Nitrospira). This study identified the number of previously understudied taxa dominating the deep aerobic freshwater habitat, suggesting that the biogeochemical cycling there is driven by the microbial community that are different from that in the epilimnion.