Understanding the influence of local and regional factors that structure biological communities can be useful in environmental conservation. Our objective was to verify whether a fish metacommunity in the Brazilian Atlantic Forest has a nonrandom structure along the longitudinal stream gradient. To do so, we applied the elements of metacommunity structure to examine fish distribution patterns at the micro-basin extent for 20 stream fish assemblages. Stream fish species were independently distributed following the Gleasonian pattern. The Gleasonian pattern suggested that the communities varied continuously over space, potentially reflecting the degree to which species tolerances overlap. The metacommunity structure may have resulted from the environmental gradient and has a high beta diversity. The upstream reaches have higher values from regional variables (confluence distance and declivity) and slower values on variables representing a local scale (temperature, conductivity, depth, and width). Knowing the type of structure and the drivers that shape a metacommunity, we suggested that ensuring the connectivity of streams is a good conservation strategy as the species move from one to another, being very dependent on the colonization source. This environmental management can affect biodiversity at local and regional scales, thus we would require devoting local conservation efforts to a large number of different reaches of streams and in a micro-basin regional scale.