This data release provides phytoplankton environmental DNA (eDNA) data from 19 sites in 12 different streams within the Milwaukee Metropolitan Sewerage District Planning Area in and around Milwaukee, WI. These streams include the Milwaukee River, the Kinnickinnic River, the Menomonee River, and more. Phytoplankton composition was determined using eDNA sequencing techniques. Data were collected from September to October of 2022, 2023, and 2024. An environmental sample and a quality assurance sample were collected at every site.