In line with the One Health concept, international organizations and the scientific community are strongly supporting the implementation of multisectoral surveillance systems in an attempt to improve the management of health hazards at the human-animal-environment interface. Such surveillance systems call for the establishment of collaboration across sectors and disciplines that must be evaluated to ensure they are appropriate and functional to produce the expected results. In this context, we have developed a tool, ECoSur (Evaluation of Collaboration for surveillance), to evaluate the organization, functioning, and functionalities of collaboration taking place in a multisectoral surveillance system. ECoSur relies on 22 attributes characterizing the organization of collaboration at the governance and operation level and nine attributes referring to core functions of collaboration to ensure the sustainable operation of an effective multisectoral surveillance system. Along with these attributes, three indexes allow for the characterization of the organization of collaboration at a macro level according to three processes: management, support, and operation. Attributes are evaluated based on the scoring of criteria that contribute to their definition. Once scores are captured in a spreadsheet, evaluation results are automatically generated. Their interpretation supports the identification of strengths and weaknesses of collaboration and the formulation of recommendations for its amelioration.