Summary Freshwater mussels are a particularly imperilled group of aquatic organisms. To date, there is limited understanding of their dietary and nutritional subsidies, including those from both autochthonous (aquatic) and allochthonous (terrestrial) sources. The aim of this study was to evaluate the relative contributions of potential nutritional resources to mussel biomass using a Bayesian mixing model (MixSIAR) for (i) bulk suspended versus benthic organic matter (OM) pools, (ii) general autochthonous versus allochthonous OM sources and (iii) specific individual OM sources. Nutritional subsidies of five species of freshwater mussels were assessed using natural abundance stable isotopes (δ13C, δ15N, δ2H) and natural radiocarbon (Δ14C). The ranges for each of the four isotopes were similar across all species and sampling dates, and mussel δ13C and δ15N values were similar to those measured in previous studies. Despite relatively low variability in this study, species and temporal differences in mussel δ15N, δ2H and Δ14C values were generally significant (P < 0.05). Based on δ13C values alone, bulk suspended OM subsidised mussel biomass to a greater extent than bulk benthic OM (c. 60 and 40% respectively). Modelling of multiple isotopes indicated that while the collective allochthonous nutritional sources accounted for a significant part (c. 33%) of mussel biomass, autochthonous materials collectively subsidised mussel tissue at about twice this level (c. 67). Significant amounts of 14C‐depleted aged carbon were incorporated into freshwater mussel tissue (mean equivalent 14C age = 1325 ± 68 years BP). Potential nutritional sources were also mostly aged, including benthic algae (1226 ± 556 years BP), zooplankton (1682 years BP), terrestrial soil (10 588 ± 4330 years BP) and suspended particulate OM (1443 ± 57 years BP). No single potential individual nutritional OM source explained the multiple isotopic values of freshwater mussel tissue, indicating that these organisms have a multi‐source and potentially opportunistic diet. Benthic algae and phytoplankton were quantitatively the most important individual OM subsidies to freshwater mussel nutrition at our study site (27 and 19% respectively). Observed seasonal shifts in freshwater mussel isotopic values were minimal, suggesting (i) that mussels may not be seasonally limited by individual nutritional resources and/or (ii) the replacement times of C, N and H in freshwater mussel tissues make detection of temporal nutritional subsidy shifts – if they occur – difficult to discern.