Aquatic fungi mediate important energy and nutrient transfers in freshwater ecosystems, a role potentially altered by widespread eutrophication. We studied the effects of dissolved nitrogen (N) and phosphorus (P) concentrations and ratios on fungal stoichiometry, elemental homeostasis, nutrient uptake and growth rate in two experiments that used (1) liquid media and a relatively recalcitrant carbon (C) source and (2) fungi grown on leaf litter in microcosms. Two monospecific fungal cultures and a multi-species assemblage were assessed in each experiment. Combining a radioactive tracer to estimate fungal production (C accrual) with N and P uptake measurements provided an ecologically relevant estimate of mean fungal C:N:P of 107:9:1 in litter-associated fungi, similar to the 92:9:1 obtained from liquid cultures. Aquatic fungi were found to be relatively homeostatic with respect to their C:N ratio (~11:1), but non-homeostatic with respect to C:P and N:P. Dissolved N greatly affected fungal growth rate and production, with little effect on C:nutrient stoichiometry. Conversely, dissolved P did not affect fungal growth and production but controlled biomass C:P and N:P, probably via luxury P uptake and storage. The ability of fungi to immobilize and store excess P may alter nutrient flow through aquatic food webs and affect ecosystem functioning.