Recent studies highlight linkages among the architecture of ecological networks, their persistence facing environmental disturbance, and the related patterns of biodiversity. A hitherto unresolved question is whether the structure of the landscape inhabited by organisms leaves an imprint on their ecological networks. We analyzed, based on pyrosequencing profiling of the biofilm communities in 114 streams, how features inherent to fluvial networks affect the co-occurrence networks that the microorganisms form in these biofilms. Our findings suggest that hydrology and metacommunity dynamics, both changing predictably across fluvial networks, affect the fragmentation of the microbial co-occurrence networks throughout the fluvial network. The loss of taxa from co-occurrence networks demonstrates that the removal of gatekeepers disproportionately contributed to network fragmentation, which has potential implications for the functions biofilms fulfill in stream ecosystems. Our findings are critical because of increased anthropogenic pressures deteriorating stream ecosystem integrity and biodiversity.