Urbanization-driven land use transitions, especially impervious surface expansion, profoundly affect aquatic ecosystems, yet the regulatory mechanisms of microbial assembly and disassembly remain unclear. Using environmental DNA metabarcoding, this study examined relationships between landscape patterns, environmental gradients, and microbial communities in the middle-lower Yangtze River. Impervious surfaces (source landscapes) increased from 10% (midstream) to 37% (downstream), disrupting source–sink balance, elevating nitrogen and phenolics, and worsening water quality. Eukaryotes exhibited higher α-diversity and more complex co-occurrence networks, showing greater responsiveness to disturbance. Stochastic processes dominated community assembly (>68%) for both eukaryotic and prokaryotic communities. Dispersal limitation led to higher total β-diversity and nestedness in eukaryotes; by contrast, land use intensified environmental filtering on prokaryotes, resulting in lower β-diversity but higher turnover contribution. Community disassembly simulation reveals a scenario in which impervious surface expansion is associated with non-random species loss, reducing predicted functional richness of both eukaryotic and prokaryotic microbes; the preferential loss of sensitive species is associated with a sharp decline in predicted functional richness, which may have implications for ecosystem stability. Our findings demonstrate that land use change is significantly associated with altered aquatic environments and affects ecosystem functions through biodiversity changes, providing theoretical support for ecosystem conservation.