In arable cropping systems, reduced or conservation tillage practices are linked with improved soil quality, C retention and higher microbial biomass, but most long-term studies rarely focus on depths greater than 15 cm nor allow comparison of microbial community responses to agricultural practices. We investigated microbial community structure in a long-term field trial (12-years, Lincoln, New Zealand) established in a silt-loam soil over four depth ranges down to 30 cm. Our objectives were to investigate the degree of homogenisation of soil biological and chemical properties with depth, and to determine the main drivers of microbial community response to tillage. We hypothesised that soil microbiological responses would depend on tillage depth, observed by a homogenisation of microbial community composition within the tilled zone. Tillage treatments were mouldboard plough and disc harrow, impacting soil to ∼20 and ∼10 cm depth, respectively. These treatments were compared to a no-tillage treatment and two control treatments, both permanent pasture and permanent fallow. Bacterial and fungal communities collected from the site were not impacted by the spatial location of sampling across the study area but were affected by physicochemical changes associated with tillage induced soil homogenisation and plant presence. Tillage treatment effects on both species richness and composition were more evident for bacterial communities than fungal communities, and were greater at depths <15 cm. Homogenisation of soil and changing land management appears to redistribute both microbiota and nutrients deeper in the soil profile while consequences for soil biogeochemical functioning remain poorly understood.