This study evaluates the sequences of biofilm colonization at two stream sites located in different bioclimatic regions (Mediterranean and Central European). Despite of the ecoregional differences, the two selected streams mainly differed by their hydrology and nutrient condition. A range of structural [microbial biomass, nutrient content, extracellular polymeric substances (EPS)-polysaccharide content] and metabolic (extracellular enzyme activities) descriptors were analyzed during the colonization period of 60 days. The succession of bacterial communities was investigated through the 16S rDNA gene analysis and taxonomical identification was used in diatom communities. Differences in algal biomass were not significant between the two stream biofilms, but the bacterial density and aminopeptidase and β-glucosidase activities were higher in the Mediterranean biofilms, probably due to greater coarse particulate organic matter (CPOM) accumulation in the latter. The colonization sequences of algae and bacteria in the biofilms were faster in the Mediterranean stream biofilms and slower and gradual in the Central European stream biofilms. Floods caused disruption in the microbial succession and re-colonization, favouring the re-appearance of early colonizing taxa and increasing the community diversity. Biofilms in the Mediterranean stream had a higher amount of early colonizing species (the diatoms Ulnaria ulna and Karayevia clevei and the β-proteobacteria group), as well as higher polysaccharide development in the extracellular polymeric substances (EPS) matrix. These differences could be an adaptation to the marked hydrological changes characteristic of Mediterranean streams. In contrast, the microbial community complexity gradually increased and nutrients and proteins largely accumulated in the Central European biofilms, reflecting the higher hydrological stability as well as the higher nutrient availability.