- The wellbeing of the Willamette basin in Oregon depends on the river ecosystem’s microbial communities, which control primary production and the biological processing of nutrients, pollutants, and organic matter. Yet, only a few studies have investigated the diversity of the microbial communities of the Willamette basin. This paper examines how prokaryotic and eukaryotic diversity of Oregon’s Willamette River Basin changes from the headwaters to the main river system. This was done by surveying microbial diversity in water samples collected at 16 sites along the Willamette River and tributaries in December 2019 and comparing them with diversity in 296 other water samples collected from across the Willamette Basin in 2017, 2018, and 2019. Categorization of species of the microbial communities at each site was identified by targeting the 18 rRNA (eukaryotic) and 16 rRNA (prokaryotic) genes. Prokaryotic community diversity varied primarily by total stream length upstream of the sampling site separating into groups associated with big (>500 km2) and small (< 500 km2) drainage basins. Within each of these groups, prokaryotic communities varied primarily by season. Analysis of the 16 representative samples illustrated the impacts of tributary inflow to the main Willamette River of both eukaryotic and prokaryotic communities. Eukaryotic populations were less affected by inflows than prokaryotic communities. Prokaryotic and eukaryotic communities showed significant correlation in small basin sites, but not in large basin locations.