Natura 2000 (N2k) is a multinational and coordinated conservation network designated to support the long-term survival of Europe's most valuable species and habitats. It is the key conservation tool in the European Union. We reviewed 510 peer-reviewed publications (period 1998–2014) focusing on ecological aspects of N2k, with the aims of identifying key research gaps and proposing future research priorities for improved conservation success. We categorized the articles by spatial scale, biogeographical regions, taxonomic groups, habitat types, and the analytical methods used. The majority of studies were performed in single N2k sites or at the regional level within countries. The Mediterranean region had the greatest number of publications and the terrestrial Black Sea, Macaronesia, Pannonian and Steppic regions were overrepresented in relation to their total area and to the area of N2k sites that they comprised. Grasslands, freshwater and wetland habitats were overrepresented in comparison to their area within N2k. Plants were the most commonly studied taxonomic group and quantitative empirical studies dominated. Future N2k research should address knowledge gaps by directing more efforts towards: 1) the Boreal region, 2) alpine, agricultural, forest and marine habitats, and 3) underrepresented taxonomic groups such as reptiles, amphibians, lichens and fungi. For enhanced evaluation and realization of the conservation potential of N2k, more studies will need to encompass large spatial scales and utilize modelling approaches to effectively address future climate and land-use changes.