Freshwater ecosystems and biodiversity are among the most threatened at global scale, but efforts for their conservation have been mostly peripheral to terrestrial conservation. For example, Natura 2000, the world's largest network of protected areas, fails to cover adequately the distribution of rare and endangered aquatic species, and lacks of appropriate spatial design to make conservation for freshwater biodiversity effective. Here, we develop a framework to identify a complementary set of priority areas and enhance the conservation opportunities of Natura 2000 for freshwater biodiversity, using the Iberian Peninsula as a case study. We use a systematic planning approach to identify a minimum set of additional areas that would help i) adequately represent all freshwater fish, amphibians and aquatic reptiles at three different target levels, ii) account for key ecological processes derived from riverscape connectivity, and iii) minimize the impact of threats, both within protected areas and propagated from upstream unprotected areas. Addressing all these goals would need an increase in area between 7 and 46%, depending on the conservation target used and strength of connectivity required. These new priority areas correspond to subcatchments inhabited by endangered and range restricted species, as well as additional subcatchments required to improve connectivity among existing protected areas and to increase protection against upstream threats. Our study should help guide future revisions of the design of Natura 2000, while providing a framework to address deficiencies in reserve networks for adequately protecting freshwater biodiversity elsewhere.