<p>The mycoheterotrophic nutritional mode, characterized by the acquisition of fungal-derived carbon by plants, has long captivated botanists and mycologists. Recent stable isotope analyses of fungal pelotons isolated from roots have advanced our understanding of this nutritional strategy; however, concerns remain regarding potential isotopic biases, particularly ¹⁵N depletion during lysis or extraction. To address these concerns, we focused on <i>Gastrodia pubilabiata</i>, a fully mycoheterotrophic orchid that associates with saprotrophic fungi. This species offers an ideal system to test whether peloton tissues reliably reflect fungal isotope signatures, as its mycorrhizal roots occasionally occur in direct contact with the fruiting bodies of its fungal partner. We measured δ¹³C and δ¹⁵N values in the aboveground tissues of <i>G. pubilabiata</i>, pelotons extracted from its roots, and fruiting bodies of the associated wood-decaying fungus <i>Cyanotrama gypsea</i>, along with neighboring autotrophic reference plants. The stable isotope analysis revealed that δ¹³C values were nearly identical between pelotons and fruiting bodies, while δ¹⁵N values were slightly higher in pelotons, indicating that peloton-derived isotopic data reliably reflect the fungal source. Moreover, the ¹³C and ¹⁵N enrichment observed in the orchid relative to the fungal fractions was broadly consistent with expected trophic-level fractionation, suggesting a predator–prey-like mode of nutrient transfer. Taken together, these findings support the validity of recently developed isotope-based approaches using extracted pelotons to represent fungal isotopic signatures, at least within this system.</p>