Understanding the coupled exchange of H<sub>2</sub>O and CO<sub>2</sub> between ecosystems and the atmosphere remains limited due to our inability to partition net fluxes into their individual source and sink components. For the Amazon rainforest, which plays an important role in the global balance of water and carbon, investigating these individual fluxes is critical given the environmental changes in recent years. Here, we apply a stable isotope-based approach to partition ecosystem-scale gas exchange from simultaneous eddy covariance measurements of H<sub>2</sub>O and CO<sub>2</sub> isotopologues. During the 2022 CloudRoots-Amazon campaign at the Amazon Tall Tower Observatory, high-frequency isotope flux measurements from 57 m were used to derive multi-day composite diurnal cycles of<em> δ</em> fluxes and ecosystem source compositions. A steady-state midday interval, constrained with independent leaf and soil isotopic observations, allowed us to coherently link the H<sub>2</sub>O and CO<sub>2</sub> isotopic states throughout the ecosystem (soil, canopy, leaf, atmosphere) using <em>δ</em><sup>18</sup>O.</p> <p>Isotopic flux partitioning indicates that transpiration accounts for 95.5 % of the net evapotranspiration (<em>ET</em>) of water at 14:00, with soil evaporation being responsible for 4.5 %. For CO<sub>2</sub>, <em>δ</em><sup>18</sup>O-based partitioning indicates that the respiration flux from the soil equals <span>−</span>44 % of the net ecosystem exchange (<em>NEE</em>), where the photosynthetic assimilation flux in turn is 144 % of <em>NEE</em>. The partitioning of <em>NEE</em> was found to be strongly dependent on the leaf intercellular-to-atmospheric CO<sub>2</sub> ratio (<em>c<sub>i/</sub>c<sub>a</sub></em>) which determines the (apparent) isotopic composition associated with photosynthetic assimilation (<em>δ<sub>P</sub></em>). This underlines how important detailed leaf and soil level measurements of isotopic compositions and leaf characteristics are for ecosystem-scale flux partitioning.