The composition of microbial communities responds to soil resource availability, and has been shown to vary with increasing depth in the soil profile. Soil microorganisms partly rely on root-derived carbon (C) for growth and activity. Roots in woody perennial systems like vineyards have a deeper vertical distribution than grasslands and annual agriculture. Thus, we hypothesized that vineyard soil microbial communities along a vertical soil profile would differ from those observed in grassland and annual agricultural systems. In a Pinot noir vineyard, soil pits were excavated to ca. 1.6–2.5m, and microbial community composition in ‘bulk’ (i.e., no roots) and ‘root’ (i.e., roots present) soil was described by phospholipid ester-linked fatty acids (PLFA). Utilization of soil taxonomy aided in understanding relationships between soil microbial communities, soil resources and other physical and chemical characteristics. Soil microbial communities in the Ap horizon were similar to each other, but greater variation in microbial communities was observed among the lower horizons. Soil resources (i.e., total PLFA, or labile C, soil C and nitrogen, and exchangeable potassium) were enriched in the surface horizons and significantly explained the distribution of soil microbial communities with depth. Soil chemical properties represented the secondary gradient explaining the differentiation between microbial communities in the B-horizons from the C-horizons. Relative abundance of Gram-positive bacteria and actinomycetes did not vary with depth, but were enriched in ‘root’ vs. ‘bulk’ soils. Fungal biomarkers increased with increasing depth in ‘root’ soils, differing from previous studies in grasslands and annual agricultural systems. This was dependent on the deep distribution of roots in the vineyard soil profile, suggesting that the distinct pattern in PLFA biomarkers may have been strongly affected by C derived from the grapevine roots. Gram-negative bacteria did not increase in concert with fungal abundance, suggesting that acidic pHs in lower soil horizons may have discouraged their growth. These results emphasize the importance of considering soil morphology and associated soil characteristics when investigating effects of depth and roots on soil microorganisms, and suggest that vineyard management practices and deep grapevine root distribution combine to cultivate a unique microbial community in these soil profiles