Understanding how spatial heterogeneity in soil microbial community structure and enzyme activities vary seasonally, and identifying the underlying mechanisms, are crucial for predicting how soil organic matter dynamics and nutrient cycling may respond to environmental change. We examined spatial variability in microbial community structure and enzyme activity and measured associated changes in plant biomass, soil microclimate, soil nutrients and other soil characteristics for 75 sampling points across a 15 × 15 m area of a Leymus chinensis meadow steppe in northeastern China over three seasons (summer, fall and spring). We observed seasonal variation in the spatial patterns of soil microbial community structure and enzyme activity, and the plant and soil variables associated with this variation also changed over time. Specifically, the microbial community structure exhibited strong spatial dependence, with high a relative spatial variance [C / (C + C0) ≥ 0.9] in summer, whereas it was more homogeneous in spring and fall. In summer, the spatial patterns of plants, soil dissolved inorganic N and dissolved organic C were the most significant predictors of spatial variation in microbial community structure. In fall, a mix of plant and soil physical explanatory variables explained 22–46 % of the spatial variation in microbial community structure. Meanwhile, only soil physical variables (soil bulk density and electrical conductivity) were strongly associated with spatial variation in microbial community structure in spring. The spatial links between microbial community structure and enzyme activity were reshaped throughout the growing season. We observed strong spatial links between soil microbial community structure and enzyme activity in spring and fall. In summer, the association between soil enzyme activities and microbial biomass was decoupled, indicating that microbial biomass showed higher turnover than soil enzymes at this time. Overall, our results reveal the drivers of spatial variation in soil microbial community structure and enzyme activity shift seasonally, highlighting the value of seasonal sampling to accurately estimate the heterogeneity and complexity of ecosystem-level processes in grasslands.