Aquatic hyphomycetes associated with leaf litter in the streambed are key to the maintenance of matter, energy fluxes, and ecosystem services of streams. Studies have shown that this community depends on environmental variables. However, the spatial scale that determines the richness and abundance of aquatic hyphomycetes is poorly understood. Thus, we aimed to evaluate the determinant spatial scale for this community. We sampled aquatic hyphomycetes and water variables from nine headwater streams in a protected area in southern Brazil. We found that the mesoscale environmental variables measured as catchment properties better explained the spatial variation in the community compositions than microscale variables. However, the microscale measured in pools within the 30 m stretch also accounted for some part of the community variation. Among the selected variables, nitrate and altitude were the main determinants on the mesoscale, while temperature, conductivity, turbidity and pH were the main determinants on the microscale. These findings suggest that each stream should be considered a single system. The characteristics of the system were found to influence these fungi more than specific variables in pools in which fungi are located, which contrasts with most studies about microbiota. Conservation actions should consider the whole stream and the potential that anthropogenic impacts may change the environmental variables throughout these ecosystems.