We investigate the "macronutrient-access hypothesis", which states that the balance between stoichiometric macronutrient demand and accessible macronutrients controls nutrient assimilation by aquatic heterotrophs. Within this hypothesis, we consider bioavailable dissolved organic carbon (bDOC), reactive nitrogen (N) and reactive phosphorus (P) to be the macronutrients accessible to heterotrophic assimilation. Here, reactive N and P are the sums of dissolved inorganic N (nitrate-N, nitrite-N, ammonium-N), soluble-reactive P (SRP), and bioavailable dissolved organic N (bDON) and P (bDOP). Previous data from various freshwaters suggests this hypothesis, yet clear experimental support is missing. We assessed this hypothesis in a proof-of-concept experiment for waters from four small agricultural streams. We used seven different bDOC:reactive N and bDOC:reactive P ratios, induced by seven levels of alder leaf leachate addition. With these treatments and a stream-water specific bacterial inoculum, we conducted a 3-day experiment with three independent replicates per combination of stream water, treatment, and sampling occasion. Here, we extracted dissolved organic matter (DOM) fluorophores by measuring excitation-emission matrices with subsequent parallel factor decomposition (EEM-PARAFAC). We assessed the true bioavailability of DOC, DON, and the DOM fluorophores as the concentration difference between the beginning and end of each experiment. Subsequently, we calculated the bDOC and bDON concentrations based on the bioavailable EEM-PARAFAC fluorophores, and compared the calculated bDOC and bDON concentrations to their true bioavailability. Due to very low DOP concentrations, the DOP determination uncertainty was high, and we assumed DOP to be a negligible part of the reactive P. For bDOC and bDON, the true bioavailability measurements agreed with the same fractions calculated indirectly from bioavailable EEM-PARAFAC fluorophores (bDOC r² = 0.96, p < 0.001; bDON r² = 0.77, p < 0.001). Hence we could predict bDOC and bDON concentrations based on the EEM-PARAFAC fluorophores. The ratios of bDOC:reactive N (sum of bDON and DIN) and bDOC:reactive P (equal to SRP) exerted a strong, predictable stoichiometric control on reactive N and P uptake (R² = 0.80 and 0.83). To define zones of C:N:P (co-)limitation of heterotrophic assimilation, we used a novel ternary-plot approach combining our data with literature data on C:N:P ranges of bacterial biomass. Here, we found a zone of maximum reactive N uptake (C:N:P approx. > 114: < 9:1), reactive P uptake (C:N:P approx. > 170:21: < 1) and reactive N and P co-limitation of nutrient uptake (C:N:P approx. > 204:14:1). The “macronutrient-access hypothesis” links ecological stoichiometry and biogeochemistry, and may be of importance for nutrient uptake in many freshwater ecosystems. However, this experiment is only a starting point and this hypothesis needs to be corroborated by further experiments for more sites, by in-situ studies, and with different DOC sources.