1) Understanding if and how trees coordinate rooting depth and aboveground hydraulic traits to define drought-resistance strategies in seasonal Amazon forests is a major gap to model parametrization aimed at predicting the effects of climate change in these ecosystems. 2) We assessed the rooting depth of 12 dominant tree species (representing ~ 42% of the forest basal area) in a seasonal Amazon forest, using the stable isotope ratios (δ18O and δ²H) of water collected from tree xylem and soils from a range of depths. We took advantage of a major ENSO-related drought in 2015/2016 that caused substantial evaporative isotope enrichment on soil. We measured the minimum dry-season leaf water potential both in a normal year (2014; Ψnon-ENSO) and in an extreme drought year (2015; ΨENSO). Furthermore, we measured xylem hydraulic traits that indicate the range of water potentials that trees tolerate without risking hydraulic failure (P50 and P88). 3) We demonstrate that coexisting trees are largely segregated along a single hydrological niche axis defined by root depth differences, access to light, and tolerance of low water potential. These differences in rooting depth were strongly related to tree size; diameter at breast height (DBH) explained 72% of the variation in the δ18Oxylem. Additionally, δ18Oxylem explained 49% of the variation in P50 and 70% of P88, with higher tolerance of low water potential in shallow-rooted species, while δ18O of xylem water explained 47% and 77% of the variation of minimum Ψnon-ENSO and ΨENSO. 4) We propose a new formulation to estimate an effective functional rooting depth, i.e., the likely soil depth from which roots can sustain water uptake for physiological functions, using DBH as predictor of root depth at this site. Based on these estimates, we conclude that a number of families, genera and species are restricted to drawing water from shallow to deep soil in a large area of the Tapajós forest. 5) Our results support the theory of hydrological niche segregation and its underlying trade-off related to drought resistance, which also affect the dominance structure of trees in this seasonal eastern Amazon forest.