Magnesium (Mg) and zinc (Zn) play important roles in biological production in stream ecosystems. Aquatic organisms take up and accumulate metals from the diet and the water; however, relative contributions are largely unknown. We determined Mg and Zn stable isotope ratios (δ26Mg, δ66Zn) of aquatic macroinvertebrates and of small goby, and their potential Mg and Zn sources (periphyton, plant litter, stream water) from upper and lower reaches of two temperate streams in the Lake Biwa catchment. Our goal was to understand Mg and Zn sources, and to explore δ26Mg and δ66Zn variations across functional groups. δ26Mg and δ66Zn did not correlate with each other indicating that Mg and Zn sources differ across feeding habitats and/or isotopic fractionation occured during Mg and Zn uptake. Grazers, shredders and predators showed offsets to higher δ26Mg and δ66Zn values compared to metal sources indicating isotope fractionation during Mg and Zn accumulation. In contrast, stream water was the main Mg and Zn source to filter-feeders as they reflected the δ26Mg and δ66Zn of stream water. Our results demonstrate the usefulness of applying non-traditional isotopes to understand metal sources to aquatic organisms, which could be used to trace the ultimate origin of metals from the catchment.