Background: According to WHO-FAO, biosecurity is “a strategic and integrated approach that encompasses the policy and regulatory frameworks (including instruments and activities) that analyse and manage risks in the sectors of food safety, public health, animal life and health, and plant life and health, including associated environmental risk”. Biosecurity is a key element of the European Union Animal Health Strategy since 2007 and is part of the recent European law on animal health in application since April 2021. Biosecurity serves the purpose of the International Health Regulation adopted in 2005 by the World Health Organization which is “to prevent, protect against, control and provide a public health response to the international spread of disease in ways that are commensurate with and restricted to public health risks, and which avoid unnecessary interference with international traffic and trade”. Biosecurity includes all measures preventing the introduction and/or spread of pathogens. As part of the One Health concept, biosecurity is particularly important as it includes the prevention of the spread to humans, to animals, to plants and to the environment. It is therefore a holistic and integrated approach, which considers also the interactions between different stakeholders and sectors. To increase the farmers’ resilience towards infectious diseases and answer the major public health challenges regarding zoonoses and antimicrobial resistance, it is important to improve the biosecurity levels in cattle farms based on cattle holders’ needs and priorities. Methodology: The study is divided into five parts. The first section aimed at identifying the most important animal and public health issues to be addressed in Belgium and related biosecurity measures (BSM). In order to consider different perspectives this first study was done by triangulating the information from a literature review of previous prioritization exercises, the Belgian laboratory databases and a veterinary survey. The second part consisted ...