Despite progress in understanding microbial biogeography of surface soils, few studies have investigated depth-dependent distributions of terrestrial microorganisms in subsoils. We leveraged high-throughput sequencing of 16S rRNA genes obtained from soils collected from the rare Charitable Research Reserve (Cambridge, Ontario, Canada) to assess the influence of depth on bacterial communities across various land-use types. Although bacterial communities were strongly influenced by depth across all sites, the magnitude of this influence was variable and demonstrated that land-use attributes also played a significant role in shaping soil bacterial communities. Soil pH exhibited a large gradient across samples and strongly influenced shifts in bacterial communities with depth and across different land-use systems, especially considering that physicochemical conditions showed generally consistent trends with depth. We observed significant ( p ≤ 0.001) and strongly correlated taxa with depth and pH, with a strong predominance of positively depth-correlated OTUs without cultured representatives. These findings highlight the importance of depth in soil biogeographical surveys and that subsurface soils harbour understudied bacterial members with potentially unique and important functions in deeper soil horizons that remain to be characterized.