Microorganisms are crucial components of aquatic ecosystems, playing key roles in biogeochemical cycles. Understanding microbial diversity and community assembly mechanisms is essential for river management and sustainable utilization of freshwater resources. However, the role of inter-microbial taxonomic group relationships in shaping community structures within high-altitude river ecosystems is unclear. This study utilizes high-throughput sequencing and bioinformatics analysis to describe the spatial dynamics of fungal and bacterial communities in the Yarlung Zangbo River at a broad environmental scale and to elucidate their community assembly mechanisms. The results indicate a significant distance-decay pattern in the fungal (p <
0.001) and bacterial (p <
0.001) communities of the Yarlung Zangbo River, with substantial differences in microbial taxonomic composition, diversity, and community structure across different regions (fungi ANOSIM R = 0.20, bacteria ANOSIM R = 0.63). Homogeneous selection predominated the community assembly of fungi (average: 67.4 %) and bacteria (average: 74.5 %) in aquatic environments. As altitude decreases, the influence of deterministic processes on fungal communities increases, while their influence on bacterial communities decreases. At the basin scale, the community structures of fungi and bacteria are mainly influenced by the degree of functional or ecological niche differentiation of another taxonomic group, as well as the hydrothermal conditions of the basin that vary with longitude. This study enhances the understanding of fungal and bacterial biogeographic patterns and community assembly mechanisms in plateau rivers, providing new perspectives for microbial ecological research in these ecosystems.