The river continuum concept predicts that gradual changes in river geomorphology and hydrology can drive longitudinal changes in aquatic community structure. Accordingly, we evaluated how environmental variables (electrical conductivity, pH, water transparency and water velocity), spatial factors (distance from headwaters and Moran’s eigen vector maps - MEMs), and the presence of dams affect small-sized fish assemblages along a 105-km stretch of the upper Branco River, a tributary in the Madeira River, Amazonas Basin, Brazil. Seine-net based collections were carried out at 15 sites up- and downriver from dams during the 2019 dry season. We captured a total of 4,330 individual fish belonging to three orders, nine families and 26 species (and a hybrid individual). Electrical conductivity and pH were affected by the presence of dams and the distance from headwaters. Species richness and abundance did not vary in response to environmental variables. While species richness showed no significant variation along the sampled river section, abundance showed a negative relationship with distance from headwaters. Species composition varied significantly in response to pH, linear spatial factors and the presence of dams. Our results suggest that change in species composition of small-sized fish assemblages in the upper Branco River occurs due to variation in water characteristics, inherent dispersal limitation, and in response to the presence of dams.