Climate change, increasing population densities, and intensified globalisation in trade, travel and migration are among the most important factors shaping the 21st century. Each impacts upon population health and the risk of infectious disease, particularly those originating at the human-animal-environmental interface. The recognition that many risk drivers of infectious disease fall outside of the typical domain of the health sector creates the challenge of identifying and pursuing priorities for cross-sectoral action aimed at strengthening global health security. In response, the One Health concept has emerged, as have related initiatives addressing Planetary Health and Biodiversity and Human Health. From a public health perspective and operationally speaking, the One Health approach offers great potential, emphasising as it does cooperation and coordination between multiple sectors. Yet despite having been a focal point for discussion for over a decade, numerous challenges facing the implementation of One Health preparedness strategies remain. While some are technical, related to the requirement for innovative early warning systems or new vaccines, for example, others are institutional and cultural in nature, given the transdisciplinary nature of the topic. There have thus been calls to address One Health from multiple perspectives, from ecology to the social sciences. In order to further explore this issue and to identify priority areas for action for strengthening One Health preparedness in Europe, ECDC convened an expert consultation on 11–12 December 2017.