Classic studies in the 1980s using 32P demonstrated the importance of leaf litter and associated microorganisms for dissolved phosphorus (P) uptake. However, the functional response of aquatic hyphomycete fungi – dominant microbial litter decomposers in forest streams – to elevated dissolved P concentrations remains poorly understood. Whole-stream nutrient-addition experiments at Coweeta Hydrologic Laboratory (North Carolina, USA) have showed that, as the percent P content of litter increases, fungal biomass increases at a slower rate, indicating ‘luxury’ storage of P. In this study, we investigated whether litter-associated fungi store excess P by conducting P-enrichment experiments that tested short-term effects of elevated dissolved P concentration on fungal biomass and litter P content. After incubating two leaf species, fast-decomposing tulip poplar (Liriodendron tulipifera) and slow-decomposing white oak (Quercus alba), in a second-order stream in the Coweeta basin, we returned at early- (day 50), mid- (day 100), and late-decay (day 150) stages and added 200 μg/L of P over one-week periods, collecting litter after 0, 4, 8, 24, 72, and 168 h of P enrichment. Litter samples were analyzed for C, N, and P content and fungal biomass (measured as ergosterol). Fungal biomass did not increase proportionally with rising fungal P content, suggesting that fungi stored excess P. To investigate this mechanism further, nuclear magnetic resonance (NMR) was used to distinguish among P-containing compounds in the litter, and estimate the mass of polyphosphate, the primary storage form of P in fungi. Polyphosphate mass was significantly higher in enriched compared to non-enriched reaches across all white oak decay stages and the early decay stage of tulip poplar. This supported the hypothesis that aquatic hyphomycetes store excess P as polyphosphate under elevated dissolved P conditions.