Tropical streams are one of the most endangered ecosystems in the world due to the constant pressures from human activities. Among these activities, agriculture represents a land use that is crucial for human development but also a key driver of stream degradation and biodiversity decline in the tropics. Against this background, we investigated indirect effects of agriculture (alterations in stream flow and nutrient availability) and climate characteristics (water temperature) on benthic biofilm communities in tropical streams (São Paulo State, Brazil). Three first-order streams draining catchments dominated by agricultural land use (sugarcane for bioenergy production, pasture) with some remaining riparian forest were studied for one year. We focused on the relationships of benthic biofilm biomass, algal biomass, diatom community and functional structure with streamflow dynamics, nitrate concentrations and water temperature. Our results indicate that these biological responses were mainly mediated by flow and water temperature and not by resource availability in the studied headwater streams. This result could be explained by the heavy rains and elevated runoff generation in these tropical catchments under agricultural influence, which might override the known effects of nutrient enrichment on benthic biofilm communities. Considering forecast climate and land-use changes in tropical streams, our findings may suggest potential shifts in benthic biofilm communities, with functional consequences for aquatic food webs in these environments.