The farming practices in vineyards vary widely, but how does this affect vineyard soils? The main objective of this study was to evaluate the effects of vineyard management practices on soil organic matter and the soil microbial community. To this end, we investigated three adjacent vineyards in the Traisen valley, Austria, of which the soils had developed on the same parent material and under identical environmental/site conditions but were managed differently (esp. tillage, fertilizer application, cover crops) for more than 10 yrs. We found that topsoil bulk density (BD) decreased with increasing tillage intensity, while subsoil BD showed the opposite trend. Soil organic carbon (SOC) stocks in 0–50 cm depth increased from 10 kg m−2 in an unfertilized and frequently tilled vineyard to 17 kg m−2 in a regularly fertilized but less intensively tilled vineyard. Topsoil microbial biomass per unit SOC, estimated by the sum of microbial phospholipid fatty acids (PLFAs), followed this trend, albeit not statistically significantly. Principal component analysis of PLFA patterns revealed that the microbial communities were compositionally distinct between different management practices. The fungal PLFA marker 18:2ω6,9 was highest in the vineyard with the lowest amount of extractable Cu (by 0.01 m CaCl2), and the bacterial-to-fungal biomass ratio was positively correlated with extractable Cu. Our results indicate that tillage and fertilizer application of vineyards can strongly affect vineyard soil properties such as BD and SOC stocks and that the application of Cu-based fungicides may impair soil fungal communities.