To compare the contributions of environmental and spatial factors in structuring assemblages of temperate stream fish on different spatial scales, I evaluated the distance decay of fish assemblage similarity and correlations among species compositions, environmental factors and geographical locations at medium (inter-reach scale, spatial extent 40km) and fine (inter-microhabitat scale, spatial extent <200m) scales. Partial redundancy analysis and variation partitioning indicated that the ordinal rank of the relative importance of environmental and spatial factors differed among scales. At the medium scale, the distance decay of similarity of species composition was steep at approximately >10-km scale, and the assemblage structure was simply explained by the distance between sites and several environmental factors (e.g., elevation and current velocity). In contrast, the distance between microhabitats explained only a small portion of the variance in species composition at the fine scale, and fish assemblages were affected by several spatial patterns of habitat (or some environmental features associated with those spatial patterns). Environmental factors at the fine scale (e.g., substratum characteristics and presence/absence of cover) correlated with each other and were spatially structured, and their contribution to species variance was smaller than that at the medium scale. These results provide evidence for scale-dependent alternation of the rank of the relative importance of environmental and spatial factors in structuring assemblages of stream fishes via the turnover of crucially contributing factors from medium to fine spatial scales.