Aquatic microbes, which are highly diverse in freshwater biofilms, play a vital role in shaping the structure and functioning of ecosystems. Nevertheless, the influence of environmental factors on aquatic microbes in different biogeographic ecosystems, especially in tropical mountains, is relatively unexplored. Here, we examined the relative influence of climate and physicochemical factors on beta diversity of stream bacteria and fungi using 16S rRNA amplicon sequencing at 40 sites on Mt. Kilimanjaro. Towards larger elevational distance, the total beta diversity and turnover of bacteria and fungi increased significantly, while nestedness decreased. The beta diversity of fungi showed greater spatial variation than that of bacteria, while the local contribution to beta diversity (LCBD) showed U-shaped elevational pattern for both taxa. Bacteria and fungi had strong positive relationships regarding the total beta diversity, LCBD and their components, except for nestedness, indicating similar environmental preferences and resource overlap. Among environmental variables, mean annual temperature, water depth and elevation predicted microbial beta diversity, while water depth, mean annual temperature and precipitation primarily affected LCBD. Overall, climate-related variables were the dominant factors for beta diversity and their effects were more pronounced for fungi than bacteria. The contrasting patterns of bacterial and fungal beta diversity and their ecological interactions in tropical aquatic ecosystems provide a rare dataset for assessing ecological functions in understudied aquatic ecosystems and predicting the impact of global change on microorganisms.