Freshwater systems are biodiversity hotspots that provide ecosystem services vital to the well-being and livelihoods of billions of people. However, the multiple stressors they face from human activity threaten their ecological integrity. This study used the Santa Ana River Watershed (SARW) in Southern California as a case study of the challenges faced by freshwater systems in semi-arid, Mediterranean-climate regions. A baseline assessment of quantitative indicators spanning ecosystem vitality (EV) and ecosystem service (ES) delivery was conducted using the Freshwater Health Index to evaluate patterns between ecosystem condition and ecosystem service delivery. Composite scores were calculated from 16 sub-indicators across EV and ES, revealing an imbalance: EV scored 40/100, while ES scored 74/100. The low EV score reflected an extensively altered flow regime, impaired water quality for native freshwater species, disrupted connectivity, and threatened freshwater biodiversity. In contrast, the high ES score reflected a robust and diversified water portfolio that meets human needs and protects cultural and recreational sites, supported in part by engineered infrastructure and imported water supplies. These results indicate an imbalance between ecosystem condition and ecosystem service delivery, consistent with patterns observed in highly managed freshwater systems. As the first basin-wide application of the FHI in a highly urbanized semi-arid Mediterranean watershed, this study can inform follow-up within-basin assessments that incorporate climate change and management scenarios to support water management actions that promote freshwater health. More broadly, this study demonstrates how freshwater health assessments can be applied in highly urbanized, semi-arid Mediterranean watersheds facing environmental and water management challenges.