This U.S. Geological Survey (USGS) Data Release provides spatial water-quality data collected from the Caloosahatchee River on June 21-22 and July 20, 2021, south Florida. Geo-referenced measurements of near surface water temperature, specific conductance, dissolved oxygen, pH, turbidity, chlorophyll fluorescence, phycocyanin fluorescence, and fluorescence of dissolved organic matter were recorded at 20 second intervals and nitrate and nitrite as nitrogen was recorded at 5 to 60 second intervals during water-quality surveys in order to create high resolution water-quality maps of the study area.