Biofilms constitute an important issue in microbial ecology, due to their high ecological and economic relevance, but the impact of abiotic conditions and microbial key players on the development and functionality of a natural biofilm is still little understood. This study investigated the effects of light intensity (LI) and bed shear stress (BSS) and the role of dominant microbes during the formation of natural biofilms and particularly the process microbial biostabilization. A comprehensive analysis of microbial biomass, extracellular polymeric substances produced, and the identification of dominant bacterial and algal species was correlated with assessment of biofilm adhesiveness/stability. LI and BSS impacted the biofilms in very different ways: biofilm adhesiveness significantly increased with LI and decreased with BSS. Moreover, microbial biomass and the functional organization of the bacterial community increased with LI, while the dynamics in the bacterial community increased with BSS. Most stable biofilms were dominated by sessile diatoms like Achnanthidium minutissimum or Fragilaria pararumpens and bacteria with either filamentous morphology, such as Pseudanabaena biceps, or a potential high capacity for extracellular polymeric-substance production, such as Rubrivivax gelatinosus. In contrast, microbes with high motility, such as Nitzschia fonticola, Pseudomonas fluorescens, and Caulobacter vibrioides, dominated the least adhesive biofilms. Their movement and potential antibiotic production could have had a disruptive impact on the biofilm matrix, which decreased its stability. This is the first study to unveil the link between abiotic conditions and resulting shifts in key microbial players to impact the ecosystem-service microbial biostabilization.