Measuring C and N stable isotopes at natural abundance can provide information on the role of fungi associated with litter decay in the nutrient cycle of freshwater ecosystems. However, uncertainty regarding isotopic fractionation by decomposer fungi during uptake or metabolic turnover is a serious limitation, weakening the description of taxon-specific ecological differences in nutrient transfer in aquatic detritus-based systems. We performed two laboratory experiments to assess C and N isotopic changes during leaf litter colonization by: (1) mixed fungal communities on three different leaf litter species, and (2) four different fungal strains growing on the same leaf litter. Our approach served to decouple the isotopic effects of different fungal taxa from those arising from structural changes occurring in leaf litter during decomposition. N isotopic changes were directly related to fungal biomass accrual on litter, whereas carbon isotopic changes were mainly dependent on the remaining lignin fraction. Isotopic mixing model equations enabled us to calculate strain-specific isotopic fractionation, indicating that fungi were enriched in 15N by 3.6–5.9‰ with respect to bulk N litter.