Phosphorus availability often limits primary production in freshwater ecosystems and excessive P inputs promote accelerated eutrophication. Microbial mechanisms may control O2-dependent uptake/release of P in stream sediments and biofilms, but specific organisms responsible for these cycles have not been identified. Polyphosphate Accumulating Organisms (PAOs) are purposely enriched in treatment plants to remove P from wastewater. PAOs release P under anaerobic conditions and take it up under aerobic conditions. It is hypothesized that alternating aerobic/anaerobic conditions promote patterns of P uptake/release similar to those attributed to PAOs in wastewater treatment. Intact, native stream biofilms were collected in the Cascadilla Creek Watershed in Tompkins County, New York, and subjected to laboratory treatments to impose conditions similar to what may occur because of diel oxygenic and respiratory cycles: 1) continuous sparging with air and 2) alternate sparging with air or anaerobic gas (20∶80% by volume CO2∶N2). PO43−, Ca, Mg, total Mn, K, Fe2+, and total S (TS) concentrations in the water were monitored and total P (TP) and polyphosphate (polyP) concentrations in the biofilms at the start and end of the experiment. Microscopy and polymerase chain reaction (PCR) were used to quantify the percentage of cells with stored intracellular polyP and to test for known PAO genes, respectively. This dataset comprises the data, analysis scripts, and visualization scripts for the paper by Saia et al. entitled 'Evidence for polyphosphate accumulating organism (PAO)-mediated phosphorus cycling in stream biofilms under alternating aerobic/anaerobic conditions. Complete dataset description including information on associated journal article, data, and data analysis R scripts can be found at https://github.com/sheilasaia/paper-p-cycling-in-stream-biofilms/tree/v1.0_for_publication. Resources in this dataset:Resource Title: Zenodo data package: sheilasaia/paper-p-cycling-in-stream-biofilms: First Public Release. File Name: Web Page, url: https://doi.org/10.5281/zenodo.242599 This is the first public release of data, analysis scripts, and visualization scripts for the paper by Saia et al. entitled 'Evidence for polyphosphate accumulating organism (PAO)-mediated phosphorus cycling in stream biofilms under alternating aerobic/anaerobic conditions' published in Freshwater Science.