The long-term reference watershed (WS4), at Fernow Experimental Forest, West Virginia, displays symptoms of N saturation despite considerable spatial heterogeneity in soil properties, such as texture, N pools, and cycling rates. We identified a weathering gradient of three sites within WS4 by assessing differences in clay content across a common geologic substrate. Across these sites (LN, MN, and HN), NO3 production rates vary significantly (low, medium, and high nitrification, respectively) and are negatively related to clay content. It is unknown whether microbial communities vary across these sites and contribute to variability in NO3 production. This study characterized soil microbial communities along this gradient and assessed factors potentially important in explaining microbial composition. We sampled mineral soil from each of the sites, analyzing for moisture, pH, organic matter, extractable N, and microbial biomass and community composition via phospholipid fatty acid (PLFA) analysis. Analysis of variance and canonical correspondence analysis indicated that microbial community composition varied among sites, with a predominance of fungal markers (18:2n6 and 18:1n9c) at the most weathered LN site and Gram negative bacteria (18:1n7c) at the less weathered MN and HN sites. Accordingly, the fungi/bacteria ratio increased in the direction of LN plots in ordination space. Correlations between measured environmental parameters and PLFA data suggest that acidic conditions and low NO3 abundance at the LN site have selected for fungal dominance, although other important factors known to exert an influence on soil microbial communities, such as differences in plant community and clay and organic matter content, may also be playing a role in determining the observed patterns.