Twenty-one stream reaches in northwestern Vermont were surveyed to assess the relative influence of local- and watershed-scale variables on stream biotic assemblages including fish, aquatic macroinvertebrates and birds. Data were collected during the summers of 2003 and 2004 and included quantitative and qualitative geomorphic and habitat assessments (local-scale) and land-use characterization and modelled annual flow and sediment loading (watershed-scale). Biotic assemblages were surveyed to capture characteristics related to abundance, diversity and composition. Principal components analysis (PCA) was used to generate sets of factors representing unique scenarios of geophysical data derived from various spatial extents within the watershed. These factors were then used as the independent variables in multiple regression models using the biotic data as the dependent variables. Forty significant models were built from the combination of the eight scenarios and 11 dependent variables. Fish assemblage diversity and composition were influenced by a combination of local-scale and watershed-scale variables; however, the qualitative local data were more predictive than the quantitative data. Local-scale data and sediment (model-derived) were important factors in building significant macroinvertebrate models. Bird abundance and species richness were best predicted using local geomorphic characteristics and the qualitative local data. Our results reinforce the concept that whereas both local- and watershed-scale variables affect stream biota, their relative influence depends upon the individual ecology of each taxon. In order to address these issues, comprehensive watershed management, restoration and conservation plans would benefit from assessments at multiple scales and from geomorphological, watershed and multitaxonomic perspectives. Copyright © 2010 John Wiley & Sons, Ltd.