Draining 31 states and roughly 3 million km2, the Mississippi River (MSR) and its tributaries constitute an essential resource to millions of people for clean drinking water, transportation, agriculture, and industry. Since the turn of the 20th century, MSR water quality has continually rated poorly due to human activity. Acting as first responders, microorganisms can mitigate, exacerbate, and/or serve as predictors for water quality, yet we know little about their community structure or ecology at the whole river scale for large rivers. We collected 16S and 18S rRNA gene amplicons and physicochemical samples from 38 MSR sites over nearly 3000 km from Minnesota to the Gulf of Mexico. These data represent the first of their kind for a major river and revealed distinct regime changes between upper and lower MSR microbial communities that corresponded to Strahler′s river order and nutrient concentrations. Within these assemblages, we identified subgroups of OTUs from the phyla Acidobacteria, Bacteroidetes, and Heterokonts that highly correlated with, and were predictive of, important eutrophication nutrients. This study offers the most comprehensive view of Mississippi River microbiota to date, establishes the groundwork for future temporal and spatial studies of river perturbations, and provides potential microbial indicators of river health.