In addition to trees and grasses, the savannas of central Brazil are characterised by a diverse herbaceous dicot flora. Here we tested whether the coexistence of a highly diversified assemblage of species resulted in stratification or strong overlap in the use of soil water resources. We measured oxygen and hydrogen isotope ratios of stem water from herbs, grasses and trees growing side by side, as well as the isotopic composition of water in soil profile, groundwater and rainfall, and predawn (Ψpd) and midday (Ψmd) leaf water potentials. We used a stable isotope mixing model to estimate vertical partitioning of soil water by the three growth forms. Grasses relied on shallow soil water (5–50 cm) and were strongly anisohydric. Ψpd and Ψmd decreased significantly from the wet to the dry season. Trees extracted water from deeper regions of the soil profile (60–120 cm) and were isohydric. Ψpd and Ψmd did not change from the wet to the dry season. Herbs overlapped with grasses in patterns of water extraction in the dry season (between 10 and 40 cm), but they took up water at soil depths intermediate (70–100 cm) to those of trees and grasses during the wet season. They showed seasonal changes in Ψpd but not in Ψmd. We conclude that vertical partitioning of soil water may have contributed to coexistence of these three growth forms and resulted in a more complex pattern of soil water extraction than the two-compartment model of soil water uptake currently used to explain the structure and function of tropical savanna ecosystems.