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Temporal dynamics create unique and often ephemeral conditions that can influence soil microbial biogeography at different spatial scales. This study investigated the relation between decimeter to meter spatial variability of soil microbial community structure, plant diversity, and soil properties at six dates from April through November. We also explored the robustness of these interactions over time. An historically unfertilized, unplowed grassland in southwest Germany was selected to characterize how seasonal variability in the composition of plant communities and substrate quality changed the biogeography of soil microorganisms at the plot scale (10 m × 10 m). Microbial community spatial structure was positively correlated with the local environment, i.e. physical and chemical soil properties, in spring and autumn, while the density and diversity of plants had an additional effect in the summer period. Spatial relationships among plant and microbial communities were detected only in the early summer and autumn periods when aboveground biomass increase was most rapid and its influence on soil microbial communities was greatest due to increased demand by plants for nutrients. Individual properties exhibited varying degrees of spatial structure over the season. Differential responses of Gram positive and Gram negative bacterial communities to seasonal shifts in soil nutrients were detected. We concluded that spatial distribution patterns of soil microorganisms change over a season and that chemical soil properties are more important controlling factors than plant density and diversity. Finer spatial resolution, such as the mm to cm scale, as well as taxonomic resolution of microbial groups, could help determine the importance of plant species density, composition, and growth stage in shaping microbial community composition and spatial patterns.