Biofilms are spatially structured ecosystems, characterized by patterns and architectural features determined by the interplay between endogenous and exogenous processes. As in many other structured ecosystems, the characterization of spatial interactions is pivotal to infer ecological functions. To date, the majority of studies have focused on pattern formation and morphology of single-species biofilm in microfluidics devices, drawing important conclusions about the physiological mechanisms mediating this phenomenon. However, the study of spatial organization of multi-species biofilms growing under conditions that resemble the native habitat has not been conducted extensively yet. The aim of this PhD thesis is the systematic investigation of spatial patterns formation in freshwater phototrophic benthic biofilms, under quasi-natural conditions. Benthic biofilms are a type of microbial community of global importance, for instance, for carbon cycling within streams. They are characterized by close metabolic interactions between autotrophic and heterotrophic microorganisms, and they are tightly shaped by the hydrodynamics of the surrounding water. We will research the formation of spatial organization in benthic biofilms with a special focus on the link between hydrodynamics, biofilm architecture, metabolism and biodiversity. We expect that this comprehensive view will permit an enhanced mechanistic understanding of the emergence of spatial organization in phototrophic benthic biofilms, as well as its ecological consequences.