Topography may affect soil microbial processes, however, the use of topographic data to model and predict the spatial distribution of soil microbial properties has not been widely reported. We studied the effect of topography on the activity of denitrifiers under different hydrologic conditions in a typical agroecosystem of the northern grasslands of North America using digital terrain modelling (DTM). Three data sets were used: (1) digital models of nine topographic attributes, such as elevation, slope gradient and aspect, horizontal, vertical, and mean land surface curvatures, specific catchment area, topographic, and stream power indices; (2) two soil environmental attributes (soil gravimetric moisture and soil bulk density); and (3) six attributes of soil microbial activity (most probable number of denitrifiers, microbial biomass carbon content, denitrifier enzyme activity, nitrous oxide flux, denitrification rate, and microbial respiration rate). Linear multiple correlation, rank correlation, circular–linear correlation, circular rank correlation, and multiple regression were used as statistical analyses. In wetter soil conditions, topographically controlled and gravity-driven supply of nutritive materials to microbiota increased the denitrification rate. Spatial differentiation of the denitrification rate and amount of denitrifying enzyme in the soil was mostly effected by redistribution and accumulation of soil moisture and soil organic matter down the slope according to the relative position of a point in the landscape. The N2O emission was effected by differentiation and gain of soil moisture and organic matter due to the local geometry of a slope. The microbial biomass, number of denitrifiers, and microbial respiration depended on both the local geometry of a slope and relative position of a point in the landscape. In drier soil conditions, although denitrification persisted, it was reduced and did not depend on the spatial distribution of soil moisture and thus land surface morphology. This may result from a reduction in soil moisture content below a critical level sufficient for transient induction of denitrification but not sufficient to preserve spatial patterns of the denitrification according to relief. Digital terrain models can be used to predict the spatial distribution of the microbial biomass and amount of denitrifying enzyme in the soil. The study demonstrated a feasibility of applying digital terrain modelling to investigate relations of other groups of soil microbiota with topography and the system ‘topography–soil microbiota’ as a whole.