Effective protection and management of groundwater‐dependent ecosystems (GDEs) are hindered by inadequate information on their locations and the condition of associated groundwater supplies. We addressed this knowledge gap by developing a methodology that uses existing datasets to locate GDEs (including groundwater‐dependent springs, lakes, rivers, wetlands, and species) and assess threats to groundwater quantity and quality. Here we report on the application of this method across the US state of Oregon. Nearly 40% of watersheds in Oregon contain two or more types of GDEs – termed “GDE clusters” – indicating the widespread importance of groundwater to ecosystems. Documented problems may underestimate the threat to ecosystems from altered groundwater supply or quality. Although documented occurrences of water‐table declines are limited, high densities of permitted wells (for irrigation or other commercial purposes) pose a threat to groundwater availability in 18% of GDE clusters. Furthermore, although only 5% of GDE clusters have known groundwater contamination, our assessment indicates that 30% of GDE clusters are threatened with groundwater contamination by nitrates, 30% by industrial chemicals, and 70% by pesticides. This initial assessment of GDEs and threats to their groundwater supply highlights the ecological importance of groundwater and the need to incorporate protection of GDEs in water management policy.