The influence of horizon position and aggregate size on bacterial and fungal community composition was determined. From nine sites, soils were collected from the top three horizon positions (H1, H2 and H3). Physical fractionation separated samples into large macroaggregate (LM,> 2000 mu m), macroaggregate (MAC,> 250 mu m), microaggregate (MIC,< 250 mu m), and silt and clay (SC, 53 mu m) fractions. In all samples, the structure of the bacterial and fungal community composition was assessed via restriction fragment length polymorphism (T-RFLP), and for the four aggregate sizes from the top two horizons positions an in-depth analysis of the bacterial community was conducted using next generation sequencing (NGS). Bacterial and fungal communities both differed between aggregate-sizes. Changes in the composition of the bacterial and fungal communities also occurred among horizon positions, with a significant interaction between aggregate size and horizon position evident for the bacterial community. Using NGS, it was shown that aggregate-size had a significant effect on the bacterial community in both horizon positions at both the phyla and family taxonomic levels. MAC and MIC significantly differed in the % relative abundance of bacterial groups, potentially indicating differing predation pressures. These results indicate that both horizon position and aggregate size support distinct microbial communities. Understanding these parameters is critical in our comprehension of the patterns of microbial diversity in soil.