P 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. We hypothesized that alternating aerobic/anaerobic conditions promote patterns of P uptake/release similar to those attributed to PAOs in wastewater treatment. We collected intact, native stream biofilms and subjected them 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 percent by volume CO2:N2). We monitored PO43-, Ca, Mg, total Mn, K, Fe2+, and total S (TS) concentrations in the water during the experiment and total P (TP) and polyphosphate (polyP) concentrations in the biofilms at the start and end of the experiment. We used microscopy and polymerase chain reaction (PCR) to quantify the percentage of cells with stored intracellular polyP and to test for known PAO genes, respectively. The water had significantly greater dissolved PO43- concentrations during anaerobic than during aerobic conditions. Ca, K, Mg, and total Mn concentrations mimicked PO43- concentrations over time, but Fe2+ and TS concentrations did not. Precipitation of Ca and Mg and reductive dissolution of Mn may have influenced P cycling. Percent microbially stored intracellular polyP was nearly three times greater in aerobic than anaerobic conditions. We did not find previously reported PAO genes in our biofilms, indicating the presence of novel polyP accumulators. Combined biotic and abiotic processes may be important in controlling short-term P cycling in stream biofilms.