Changed spatial configurations at sowing have been investigated as a strategy to minimize interspecific competition and improve the establishment and persistence of multi-species plantings in pastures, but the impact of this practice on the soil microbiome has received almost no previous research attention. Differences in populations of bacteria and fungi in the surface 10 cm of soil in the third year following pasture establishment were quantified using quantitative polymerase chain reaction and terminal restriction fragment length polymorphism methods. Populations were compared on, and between, drill rows sown to either the perennial grass phalaris (Phalaris aquatica L.), perennial legume lucerne (alfalfa; Medicago sativa L.) or the annual legume subterranean clover (Trifolium subterraneum L.). Results showed that soil microbial abundance and diversity were related to plant distribution across the field at the time of sampling and to soil chemical parameters including total carbon (C), mineral nitrogen (N), pH, and available phosphorus (P), potassium (K) and sulfur (S). Despite the 27-month lag since sowing, pasture species remained concentrated around the original drill row with very little colonization of the inter-row area. The abundance and diversity of bacterial and fungal populations were consistently greater under drill rows associated with higher total C concentrations in the surface soil compared with the inter-row areas. Our results showed that the pH and available nutrients were similar between the subterranean clover drill row and the inter-row, suggesting that soil microbial populations were not impacted directly by these soil fertility parameters, but rather were related to the presence or absence of plants. The abundance of bacteria and fungi were numerically lower under phalaris rows compared to rows sown to legumes. The richness and diversity of fungal populations were lowest between rows where lucerne was planted. Possible explanations for this observation include a lower C:N ratio of lucerne roots and/or a lack of fibrous roots at the soil surface compared to the other species, illustrating the influence of contrasting plant types on the soil microflora community. This study highlights the enduring legacy of the drill row on the spatial distribution of plants well into the pasture phase of a cropping rotation and discusses the opportunity to enhance the microbiome of cropping soils on a large scale during the pasture phase by increasing plant distribution across the landscape.