A geostatistical approach using replicated grassland sites (10 m × 10 m) was applied to investigate the influence of grassland management, i.e. unfertilized pastures and fertilized mown meadows representing low and high land-use intensity (LUI), on soil biogeochemical properties and spatial distributions of ammonia-oxidizing and denitrifying microorganisms in soil. Spatial autocorrelations of the different N-cycling communities ranged between 1.4 and 7.6 m for ammonia oxidizers and from 0.3 m for nosZ-type denitrifiers to scales >14 m for nirK-type denitrifiers. The spatial heterogeneity of ammonia oxidizers and nirS-type denitrifiers increased in high LUI, but decreased for biogeochemical properties, suggesting that biotic and/or abiotic factors other than those measured are driving the spatial distribution of these microorganisms at the plot scale. Furthermore, ammonia oxidizers (amoA ammonia-oxidizing archaea and amoA ammonia-oxidizing bacteria) and nitrate reducers (napA and narG) showed spatial coexistence, whereas niche partitioning was found between nirK- and nirS-type denitrifiers. Together, our results indicate that spatial analysis is a useful tool to characterize the distribution of different functional microbial guilds with respect to soil biogeochemical properties and land-use management. In addition, spatial analyses allowed us to identify distinct distribution ranges indicating the coexistence or niche partitioning of N-cycling communities in grassland soil.