In this commentary, we summarize and build upon discussions that emerged during the workshop Isotope-based studies of water partitioning and plant–soil interactions in forested and agricultural environments held in San Casciano in Val di Pesa, Italy, in September 2017. Quantifying and understanding how water cycles through the Earth's critical zone is important to provide society and policymakers with the scientific background to manage water resources sustainably, especially considering the ever-increasing worldwide concern about water scarcity. Stable isotopes of hydrogen and oxygen in water have proven to be a powerful tool for tracking water fluxes in the critical zone. However, both mechanistic complexities (e.g. mixing and fractionation processes, heterogeneity of natural systems) and methodological issues (e.g. lack of standard protocols to sample specific compartments, such as soil water and xylem water) limit the application of stable water isotopes in critical-zone science. In this commentary, we examine some of the opportunities and critical challenges of isotope-based ecohydrological applications and outline new perspectives focused on interdisciplinary research opportunities for this important tool in water and environmental science.