Regional variation in grape and wine quality characteristics is a critical feature of perceived product identity, with significant consequences for consumer preference and economic appreciation. Soil is a fundamental factor of viticulture, but the associated soil microorganisms have been largely ignored. In this study, we comprehensively investigated the microbial ecology of vineyard soils across southern Australia, suggesting soil-borne microbiome distinguish wine regions, especially fungal communities. Soils were collected from 14 vineyards (three sites per vineyard) in seven wine producing regions across southern Australia and also extensively sampled from one region to test the impact of vintage on soil microbial patterns. Next-generation sequencing of 16S ribosomal RNA and internal transcribed spacer ribosomal sequence was performed to build both bacterial and fungal distribution patterns. Results show that soil bacterial communities were dominated by Actinobacteria (32%) and Proreobacteria (31%), while fungal communities were less diverse, dominated by Ascomycota (70%). Species richness (α-diversity) of bacteria varied with regions but not fungi. Meanwhile, phylogenetic β-diversity of both bacterial and fungal communities segregates by region, based on principal coordinates analysis (PCoA) of weighted UniFrac distances. Random forest supervised learning models revealed that fungal models display a large degree of predictive power to distinguish soil samples coming from various regions. Furthermore, bacterial and fungal communities exhibit different responses to vintage and site-specific effects. Macroregion mainly drive vineyard soil bacterial community assembly, but fungi are more important for vintage effects. These findings will enrich understanding of vineyard microbial ecology, which is of great importance for regional origin of wine and agricultural systems and will enhance agricultural management and improve supply, consumer acceptance, and economic value.