The oxygen isotope composition (δ18O) of atmospheric CO2 is among a very limited number of tools available to constrain estimates of the biospheric gross CO2 fluxes, photosynthesis and respiration at large scales. However, the accuracy of the partitioning strongly depends on the extent of isotopic disequilibrium between the signals carried by these two gross fluxes. Chamber-based field measurements of total CO2 and CO18O fluxes from foliage and soil can help evaluate and refine our models of isotopic fractionation by plants and soils and validate the extent and pattern of isotopic disequilibrium within terrestrial ecosystems. Owing to sampling limitations in the past, such measurements have been very rare and covered only a few days. In this study, we coupled automated branch and soil chambers with tuneable diode laser absorption spectroscopy techniques to continuously capture the δ18O signals of foliage and soil CO2 exchange in a Pinus pinaster Aït forest in France. Over the growing season, we observed a seasonally persistent isotopic disequilibrium between the δ18O signatures of net CO2 fluxes from leaves and soils, except during rain events when the isotopic imbalance became temporarily weaker. Variations in the δ18O of CO2 exchanged between leaves, soil and the atmosphere were well explained by theory describing changes in the oxygen isotope composition of ecosystem water pools in response to changes in leaf transpiration and soil evaporation.