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Abstract The spatial heterogeneity of soil’s microhabitats warrants the study of ecological patterns and community assembly processes in the context of physical disturbance that disrupts the inherent spatial isolation of soil microhabitats and microbial communities. By mixing soil at various frequencies in a 16-week lab incubation, we explored the effects of physical disturbance on soil bacterial richness, community composition, and community assembly processes. We hypothesized that well-mixed soil would harbor a less rich microbial community, with community assembly marked by homogenizing dispersal and homogeneous selection. Using 16S rRNA gene sequencing, we inferred community assembly processes, estimated richness and differential abundance, and calculated compositional dissimilarity. Findings supported our hypotheses, with > 20% decrease in soil bacterial richness in well-mixed soil. Soil mixing caused communities to diverge from unmixed controls (Bray–Curtis dissimilarity; 0.75 vs. 0.25), while reducing within-group heterogeneity. Our results imply that the vast diversity observed in soil may be supported by spatial heterogeneity and isolation of microbial communities, and also provide insight into the effects of physical disturbance and community coalescence events. By isolating and better understanding the effects of spatial heterogeneity and disconnectivity on soil microbial communities, we can better extrapolate how anthropogenic disturbances may affect broad soil functions.