Diatoms (Division Bacillariophyta) commonly dominate benthic algal communities in upland streams and are the major primary producers that sit at the base of food webs. Given their ecological importance, the aim of the present study was to investigate the spatial patterns of freshwater biofilm biomass and diatom assemblage composition in response to differences in physical and chemical variables in the Bremer, Logan and Albert catchments in south-east Queensland. Patterns in benthic diatom assemblage structure were examined during base flow conditions and the focus was on relating the spatial hierarchical interrelationships among ultimate (i.e. catchments and geology) and proximate (i.e. flow, nutrients, light, conductivity, pH and turbidity) environmental drivers. The spatial difference observed in the benthic diatom assemblages was predominantly driven by proximate environmental drivers, which, in turn, were affected by ultimate drivers. Flow played a critical role in controlling both biofilm biomass and affecting the occurrence of diatom taxa. Nutrients and light also played roles as limiting resources structuring biofilm biomass and the relative abundance of diatoms. Diatom growth form, cell size and attachment mode can be used to create a more quantitative and predictive approach to establishing relationships between diatoms and environmental gradients.