Microbial communities represent the largest biodiversity pool in streams and drive the majority of biogeochemical cycles in this ecosystem. However, the various factors affecting bacterial assemblages are largely unexplored including the simultaneous impacts of seasonal and temporal variation. This study investigate potential factors of variations driving change in the stream biofilm bacterial biofilm communities. We used 16S rRNA gene amplicon sequencing to define community composition and to disentangle the relationships between stream biofilm communities, comprehensive water quality data and ecological and regional environmental information over 2-years. Stream biofilm samples were collected from three different catchment types (urban, rural and native streams) based on their impacts from 2013-2015. To quantify the factors affecting stream bacterial communities, we applied several multivariate statistical analyses. We found that monthly variation in bacterial community composition within streams were far more important (PERMANOVA; p=0.007) than the differences observed among different streams and catchment types (PERMANOVA; p=0.44). Bacterial community composition over months and years followed a discernible cyclical seasonal pattern as quantified using periodic regression analysis. Consideration of environmental factors using conditional inference trees confirmed that air and water temperature, dissolved oxygen, ammonia, total phosphorus were the factors most closely associated with differences in community structure. Overall, community structure is driven by changes in local physical and chemical conditions throughout the year. Collectively, this study significantly advances our understanding of the successional patterns of the stream bacterial communities exposed to different land use impacts, the ecosystem services they mediate, and likely responses to change such as restoration efforts and land use alteration.