Forest canopies alter the amount and isotopic composition of precipitation reaching the forest floor. Thus retention, evaporation and transport processes in forest canopies, and their effects on water isotopes, are key to understanding forest water cycling. Using a two-year isotope dataset from a mixed beech/spruce forest in Zurich, Switzerland, we assessed the isotopic offsets between precipitation, throughfall and stemflow. We also analysed how these offsets affect estimates of the fraction of soil water that is derived from winter precipitation. Throughfall was typically enriched in heavy isotopes compared to precipitation, but isotopically lighter than stemflow, with average δ²H of −64.3 ‰, −59.9 ‰ and − 56.3 ‰ in precipitation, throughfall and stemflow, respectively. The differences between beech and spruce were rather small compared to the seasonal differences in precipitation isotopes. Isotopic offsets between precipitation and throughfall/stemflow were smaller during the spring and summer months (March through August) than during fall and winter (September through February). Bulk and mobile soil waters at 10 and 40 cm showed smaller seasonal variations than those in precipitation, throughfall and stemflow, and were isotopically lighter than recent precipitation, with the largest offsets occurring during the summer months (June through August) for bulk soil waters. Thus, bulk soil waters at both depths contain a mixture of precipitation from previous events and seasons, with over-representation of isotopically lighter winter precipitation. Mobile soil waters were more similar to recent precipitation than bulk soil waters were. Throughfall isotopes were slightly heavier than precipitation isotopes, resulting in different sinusoidal fits for seasonal isotopic cycles in precipitation and throughfall. These differences lead to small underestimates in the fraction of soil water originating from winter precipitation, when open-field precipitation rather than throughfall is used as the input data. Together our results highlight the importance of isotope measurements in throughfall and stemflow for the assessment of precipitation seasonality and water cycling across forested landscapes.