A fundamental premise of the Millennium Ecosystem Assessment is that biodiversity and ecosystem services are key determinants of long-term sustainability of social-ecological systems. With a continuing decline in local and global biodiversity and ecosystem services, it is crucial to understand how biodiversity and various ecosystem services interact and how land change may modify these interactions over time. However, few studies have been conducted to quantify these relationships. In this study, we present the first empirical comparative results to analyze how spatial associations between biodiversity and ecosystem services (BES) changed at multiple scales between 1984 and 2010 in the rapidly urbanizing San Antonio River Basin (SARB), Texas, USA. We found statistically significant positive spatial associations among biodiversity, carbon storage, and sediment retention both in the entire SARB and the urban watersheds in Bexar County. Overall, biodiversity and carbon storage declined across the SARB, while sediment retention remained relatively stable. Moreover, the rates of biodiversity loss and carbon storage degradation were negatively related to the urban expansion and have accelerated since the inception of the North American Free Trade Agreement (NAFTA) in 1994. During the pre- and post-NAFTA periods (1984-1995 and 1995-2010, respectively) the rates of biodiversity loss increased from 0.7% to 0.9%, and the rates of carbon-storage loss increased from 0.1% to 1.4% per annum in the urban watersheds. Our hotspot analyses indicate that the upstream watersheds in the Basin, which supply water to the critically important Edwards Aquifer, should be targeted for priority conservation to mitigate the adverse impacts of land change on BES. Our results suggest the strong need for green infrastructure policies that integrate biodiversity conservation and sustainable use of multiple ecosystem services to address the environmentally deleterious impacts of the extensive land change under the NAFTA and to ensure the long-term social-ecological sustainability of the rapidly urbanizing SARB.