Although soil structure largely determines energy flows and the distribution and composition of soil microhabitats, little is known about how microbial community composition is influenced by soil structural characteristics and organic matter compartmentalization dynamics. A UV irradiation-based procedure was developed to specifically isolate inner-microaggregate microbial communities, thus providing the means to analyze these communities in relation to their environment. Whole- and inner-microaggregate fractions of undisturbed soil and soils reclaimed after disturbance by surface coal mining were analyzed using 16S rDNA terminal restriction fragment polymorphism (T-RFLP) and sequence analyses to determine salient bacterial community structural characteristics. We hypothesized that inner-microaggregate environments select for definable microbial communities and that, due to their sequestered environment, inner-microaggregate communities would not be significantly impacted by disturbance. However, T-RFLP analysis indicated distinct differences between bacterial populations of inner-microaggregates of undisturbed and reclaimed soils. While both undisturbed and reclaimed inner-microaggregate bacterial communities were dominated by Actinobacteria, undisturbed soils contained only Actinobacteridae, while in inner-microaggregates of reclaimed soils Rubrobacteridae predominate. Spatial stratification of division-level lineages within microaggregates was also seen. The fractionation methods employed in this study therefore represent a valuable tool for defining relationships between biodiversity and soil structure.