Knowledge of the degradation extent and pathways of fungicides in the environment is scarce. Fungicides may have isomers with distinct fungal-control efficiency, toxicity and fate in the environment, requiring specific approaches to follow up the degradation of individual isomers. Here we examined the degradation of the widely used fungicide dimethomorph (DIM) in a vineyard catchment using ratios of carbon stable isotopes (δ13C) and E/Z isomer fractionation (IF(Z)). In a microcosm laboratory experiment, DIM degradation half-life in soil was 20 ± 3 days, and was associated with significant isomeric (ΔIF(Z) = +30%) and isotopic (Δδ13C up to 7‰) fractionation. This corresponds to an isomer enrichment factor of εIR = −54 ± 6%, suggesting isomer selectivity and similar carbon stable isotopic fractionation values of εDIM-(Z) = −1.6 ± 0.2‰ and εDIM-(E) = −1.5 ± 0.2‰. Isomeric and isotopic fractionation values were used to estimate DIM degradation in topsoil and transport in a vineyard catchment over two wine-growing seasons. DIM concentrations following DIM application were up to 3 μg g−1 in topsoil and 29 μg L−1 in runoff water at the catchment outlet. Accordingly, the IF(Z) and δ13C values of DIM in soil were similar to those observed in DIM commercial formulations. The gradual enrichments in DIM-(Z) and 13C of the residual DIM in soil indicated DIM biodegradation over time. DIM biodegradation estimated based on E/Z isomer and carbon stable isotope ratios in topsoil and runoff water ranged from 0% after DIM application up to 100% at the end of the wine-growing season. DIM biodegradation was overestimated compared to conventional approaches relying on DIM mass balance, field concentrations and half-lives. Altogether, our study highlights the usefulness of combining carbon stable isotopes, E/Z isomers and classical approaches to estimate fungicide degradation at the catchment scale, and uncovers difficulties in using laboratory-derived values in field studies.