Freshwater ecosystems are exposed to multiple stressors, but their individual and combined effects remain largely unexplored. Here, we investigated the response of stream biofilm bacterial communities to warming, hydrological stress and pesticide exposure. We used 24 artificial streams onto which epilithic (growing on coarse sediments) and epipsammic (growing on fine sediments) stream biofilms were maintained. Bacterial community composition and estimated function of biofilms exposed during 30 days to individual and combined stressors were assessed using 16S rRNA gene metabarcoding. Among the individual effects by stressors, hydrological stress (i.e. a simulated low-flow situation) was the most relevant, as it significantly altered 57% of the most abundant bacterial taxa (n = 28), followed by warming (21%) and pesticide exposure (11%). Regarding the combined effects, 16% of all stressor combinations resulted in significant interactions on bacterial community composition and estimated function. Antagonistic responses prevailed (57-89% of all significant interactions), followed by synergisms (11-43%), on specific bacterial taxa, indicating that multiple-stressor scenarios could lead to unexpected shifts in the community composition and associated functions of riverine bacterial communities. Freshwater ecosystems such as rivers are of crucial importance for human well-being. However, human activities result in many stressors (e.g. toxic chemicals, increased water temperatures, and hydrological alterations) co-occurring in rivers and streams worldwide. Among the many organisms inhabiting rivers and streams, bacteria are ecologically crucial; they are placed at the base of virtually all food webs and they recycle the organic matter needed for bigger organisms. Most of these bacteria are in close contact with river substratum, where they form the biofilms. There is an urgent need to evaluate the effects of these stressors on river biofilms, so we can anticipate future environmental problems. In this study, we experimentally exposed river biofilms to a pesticide mixture, an increase in water temperature and a simulated low-flow condition, in order to evaluate the individual and joint effects of these stressors on the bacterial community composition and estimated function.