Abstract Carbon dioxide sequestration from the atmosphere is commonly assessed using the eddy covariance (EC) method. Its net flux signal can be decomposed into gross primary production (GPP) and ecosystem respiration components, but these have seldom been tested against independent methods. In addition, EC lacks the ability to partition carbon sequestration among individual trees or species within mixed forests. Therefore, we compared GPP from EC to an independent method based on sap flow and water-use efficiency, as measured by the tissue heat balance method and δ13C of phloem contents, respectively. The latter measurements were conducted on individual trees throughout a growing season in a mixed broadleaf forest dominated by three tree species, namely English oak, narrow-leaved ash, and common hornbeam. In this context, we applied an alternative ecophysiological method aimed at verifying the accuracy of a state-of-the-art EC system while also offering a solution to the partitioning problem. We observed strong agreement in the ecosystem GPP estimates (R2 = 0.56; p < 0.0001), with correlation being especially high and nearly on the 1:1 line in the period before end of July (i.e., DOY 212; R2 = 0.85; p < 0.0001). After this period, the estimates of GPP began to diverge. Possible reasons for the divergence are discussed, focusing especially on phenology and the limitation of the isotopic data. English oak showed the highest per-tree daily photosynthetic rates among tree species, but the smaller, more abundant common hornbeam contributed most to the stand-level summation, especially early in the spring. These findings provide a rigorous test of the methods and the species-level photosynthesis offers avenues for enhancing forest management aimed at carbon sequestration.