Lake Powell retains most of the phosphorus that it receives, leading to downstream phosphorus limitation. These data were compiled to examine controls on phosphorus cycling below Lake Powell in the Colorado River and from storm inputs from the Paria River. Objectives of our study were to determine how several forms of phosphorus, both organic and inorganic, were cycled under varying dissolved oxygen concentrations and pH, reflecting the range of values observed in the river over the years. These data represent nitrogen, phosphorus, calcium, and carbon concentrations, water quality parameters (pH, dissolved oxygen, temperature), sediment composition, total protein, and extracellular enzyme activity (alkaline phosphatase). Additionally, these data contain some previously unpublished longer term continuous pH data from the Colorado River. These data were primarily collected in the summer of 2021, before, during, and immediately following incubations of three different sediment types with Colorado river water. Sediment and overlying water for incubations were collected at one time point from three sites: the Paria River near the confluence with the Colorado River, the Colorado River approximately 23 river kilometers below Glen Canyon Dam, and the Colorado River near its inflow to Lake Mead at the Pearce Ferry boat ramp. Data were collected by the Southwest Biological Science Center-Grand Canyon Monitoring and Research Center researchers. These ambient river water and sediment data can be used to describe chemical and biological conditions in the river and data from bottle incubations can be used to examine how changing laboratory conditions affect nutrient availability and cycling.