River managers in Australia are managing in the face of extremes to provide security of water supply for people, production and the environment. Balancing the water requirements of people, environments and economies requires that water security is viewed holistically, not just in terms of the water available for human consumption. Common definitions of water security focus on the needs of both humans and ecosystems for purposes such as drinking, agriculture and industrial use, and to maintain ecological values. Information about achieving water security for the environment or ecological purposes can be a challenge to interpret because the watering requirements of key ecological processes or assets are not well understood, and the links between ecological and human values are often not obvious to water users. Yet the concepts surrounding river health are inherently linked to holistic concepts of water security. The measurement of aquatic biota provides a valuable tool for managers to understand progress toward achieving ecological water security objectives. This paper provides a comprehensive review of the reference condition approach to river health assessment, using the development of the Australian River Assessment System (AUSRIVAS) as a case study. We make the link between the biological assessment of river health and assessment of ecological water security, and suggest that such an approach provides a way of reporting that is relevant to the contribution made by ecosystems to water security. The reference condition approach, which is the condition representative of minimally disturbed sites organized by selected physical, chemical, and biological characteristics, is most important for assessing ecological water security objectives.