Sources of water use by 10 alluvial trees in various hydrogeological and ecological situations at the Pfyn forest (Wallis canton, Switzerland) were assessed by analysing 18O and 2H isotopes of precipitation, soil water at different depths, surface water, groundwater and xylem sap. The soil water line in a δ18O versus δ2H diagram shows evidence of kinetic fractionation related to evaporation. The tree water line is close to the soil trend; however, an additional enrichment may occur and could be related to xylem–phloem communication under water stress. At sites where the water table was at least 2 m below the ground surface, isotopic temporal variability of soils and trees was strongly linked with seasonal variation of soil water content. When soil water content was low and water table shallow, trees used both soil water and groundwater. When soil water content was high, this source was usually the dominant source for transpiration. In addition, some ecological strategies, reproduction or growth competition, could explain shifts in the utilization of different water sources, for example, from soil water to a mix of soil water and groundwater. At one site where soil water and groundwater were abundant throughout the year (next to the river course), neighbouring trees permanently used distinct water sources. This is consistent with a strategy of competition limitation, which would favour bank colonization. These results provide insight into the ecohydrological functioning of this system and will aid future management responses to both local and climate changes.