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Dissolved organic matter (DOM) is a major source of macronutrients to freshwaters, yet it has variable and poorly understood bioavailability. Because intrinsic variation in bioavailability is caused by chemical structures of organic nutrients, DOM composition data should improve predictions of bioavailable resource pool sizes. We hypothesized that bioavailable organic carbon (C) and nitrogen (N) fractions are made up of freshly produced humic- and protein-like DOM, respectively, whereas bioavailable phosphorus (P) is linked to microbially-derived DOM with potential organophosphate content and/or to humic-like structures associated with DOM-Fe-phosphate complexes. These hypotheses were tested from surface waters collected at eight, unproductive and organic matter-rich stream and lake sites, from which we performed C, N and P microbial bioassays with flow cytometry in combination with analyses of DOM composition using fluorescence excitation-emission matrix (EEM) analysis. Bioavailable C followed the predicted patterns, with strong links to fluorescent features indicating recently produced DOM. Surprisingly, bioavailable N was relatively poorly related to DOM composition, including protein-like fluorescence, and was instead driven mainly by the amount of inorganic N. The bioavailable P showed patterns in support of the hypothesized link to microbially-derived organic components, whereas its relationships to free or complex-bound forms of inorganic phosphate were inconclusive. Thus, the strength of the hypothesized patterns varied. Nonetheless, in addition to the variability in bioavailable nutrient concentrations explained by standard bulk nutrient variables, we show that DOM composition variables made significant and unique contributions to explaining the variance in bioavailable C (19%), N (13%) and P (18%). Therefore, improved regression models for bioavailable nutrient concentrations could be achieved by including DOM composition among the explanatory variables. Overall, DOM composition analysis is a promising tool to improve prediction and develop our understanding of bioavailable macronutrients in organic matter-rich freshwaters.