Little information is available regarding the landscape-scale distribution of microbial communities and its environmental determinants. However, a landscape perspective is needed to understand the relative importance of local and regional factors and land management for the microbial communities and the ecosystem services they provide. In the most comprehensive analysis of spatial patterns of microbial communities to date, we investigated the distribution of functional microbial communities involved in N-cycling and of the total bacterial and crenarchaeal communities over 107 sites in Burgundy, a 31 500 km2 region of France, using a 16 × 16 km2 sampling grid. At each sampling site, the abundance of total bacteria, crenarchaea, nitrate reducers, denitrifiers- and ammonia oxidizers were estimated by quantitative PCR and 42 soil physico-chemical properties were measured. The relative contributions of land use, spatial distance, climatic conditions, time, and soil physico-chemical properties to the spatial distribution of the different communities were analyzed by canonical variation partitioning. Our results indicate that 43–85% of the spatial variation in community abundances could be explained by the measured environmental parameters, with soil chemical properties (mostly pH) being the main driver. We found spatial autocorrelation up to 739 km and used geostatistical modelling to generate predictive maps of the distribution of microbial communities at the landscape scale. The present study highlights the potential of a spatially explicit approach for microbial ecology to identify the overarching factors driving the spatial heterogeneity of microbial communities even at the landscape scale.