Although international health agencies encourage the development of One Health (OH) surveillance, many systems remain mostly compartmentalized, with limited collaborations among sectors and disciplines. In the framework of the OH European Joint Programme “MATRIX” project, a generic evaluation tool called OH-EpiCap has been developed to enable individual institutes/governments to characterize, assess and monitor their own OH epidemiological surveillance capacities and capabilities. The tool is organized around three dimensions: organization, operational activities, and impact of the OH surveillance system; each dimension is then divided into four targets, each including four indicators. A semi-quantitative questionnaire enables the scoring of each indicator, with four levels according to the degree of satisfaction in the studied OH surveillance system. The evaluation is conducted by a panel of surveillance representatives (during a half-day workshop or with a back-and-forth process to reach a consensus). An R Shiny-based web application facilitates implementation of the evaluation and visualization of the results, and includes a benchmarking option. The tool was piloted on several foodborne hazards (i.e., Salmonella, Campylobacter, Listeria ), emerging threats (e.g., antimicrobial resistance) and other zoonotic hazards (psittacosis) in multiple European countries in 2022. These case studies showed that the OH-EpiCap tool supports the tracing of strengths and weaknesses in epidemiological capacities and the identification of concrete and direct actions to improve collaborative activities at all steps of surveillance. It appears complementary to the existing EU-LabCap tool, designed to assess the capacity and capability of European microbiology laboratories. In addition, it provides opportunity to reinforce trust between surveillance stakeholders from across the system and to build a good foundation for a professional network for further collaboration.