In past decades, urban tree biosecurity has taken on growing importance worldwide. Stakeholders play a key role in countering the spread of invasive alien pests and pathogens that affect the health of urban green infrastructures. The aim of this study was to increase the understanding of the role of stakeholders’ perceptions, priorities and networks in the implementation of actions to guarantee a coherent biosecure system. The research was done in three steps. First, stakeholder mapping was carried out to identify relevant actors. Second, a questionnaire on perceptions, actions and collaboration among stakeholders involved in urban biosecurity was developed and administered. Third, data was processed to compare the responses given by the different categories of stakeholders. Stakeholder mapping identified 953 relevant stakeholders of which 255 stakeholders from 19 countries completed the online survey. According to the stakeholders, the current most important urban tree pests across Europe are Cameraria ohridella and Cydalima perspectalis, mentioned in 13 and 12 countries respectively, while other pests and pathogens have been specifically mentioned in individual countries (Thaumetopoea pityocampa in Spain, Ips typographus in Latvia and Serbia, Ceratocystis platani in Italy and Türkiye). Regarding future threats to urban trees, the stakeholders emphasised significant concerns around the bacterial plant pathogen Xylella fastidiosa and insect pest Agrilus planipennis, mentioned in 12 and 14 countries respectively. In addition, the outcome of the study highlighted that the most widely adopted biosecurity actions by stakeholders are those related to communication, both to the general public (information) and to the staff involved in the biosecurity sector (training). A network analysis of relationships between stakeholder groups evidenced positive collaborations that tended to be more common the closer to hands-on practice of tree care the stakeholders were. The study provided a snapshot of the European stakeholders’ perceptions and readiness to implement biosecurity actions, highlighting the importance of connectedness as the basis to guarantee a coherent biosecure system.