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Non-traditional stable isotopes of metals were recently shown as new dietary tracers in terrestrial and marine mammals. Whether these metal stable isotopes can be used to understand feeding habits in stream food webs is not known yet. In this study, we explored the potential of stable isotopes of essential Mg (δ²⁶Mg) and Zn (δ⁶⁶Zn) as a new tool in stream ecology. For this purpose, we determined δ²⁶Mg and δ⁶⁶Zn values of stream organisms and their potential metal sources in upper and lower reaches of two streams in the Lake Biwa catchment, Central Japan. Our goals were (1) to explore variations in δ²⁶Mg and δ⁶⁶Zn across organisms of different feeding habits and (2) to understand Mg and Zn sources to stream organisms. Overall, δ²⁶Mg and δ⁶⁶Zn values of organisms were neither related to each other, nor to δ¹³C and δ¹⁵N values, indicating different elemental sources and factors controlling isotopic fractionation depending on element and taxa. Low δ²⁶Mg values in filter-feeding caddisfly larvae and small gobies indicated aqueous Mg uptake. Higher δ²⁶Mg values in leaf-shredding crane fly and grazing mayfly larvae suggested Mg isotopic fractionation during Mg uptake from the diet. While the δ²⁶Mg values of stonefly nymphs reflected those of caddisfly larvae as a potential prey, the highest δ²⁶Mg values found in dobsonfly nymphs can be explained by ²⁶Mg enrichment during maturing. δ⁶⁶Zn values of caddisfly and mayfly larvae indicated Zn was a mixture of aqueous and dietary available Zn, while higher δ⁶⁶Zn values in crane fly larvae pointed to Zn isotopic fractionation during Zn uptake from plant litter. δ⁶⁶Zn values in stonefly and dobsonfly nymphs were often in the range of those of caddisfly larvae as their prey, while dragonfly nymphs and small goby were depleted in ⁶⁶Zn relative to their dietary Zn sources. We conclude that δ²⁶Mg is a promising indicator to assess Mg sources in stream ecology depending on taxa, while the use of δ⁶⁶Zn is limited due to the complexity in Zn sources.