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Summary The anthropogenic spread of disease from captive to wild amphibian populations (referred to as spillover) is linked to global amphibian declines. Disinfecting procedures and protocols exist to mitigate pathogen transmission to and within natural areas, but understanding of visitor attitudes and behaviour regarding their adoption is limited. We surveyed visitors in two natural areas in a global amphibian biodiversity hotspot to assess their attitudes regarding pathogen spread in such areas and analysed the factors influencing their behavioural intentions to take specific actions to prevent pathogen spillover. Visitors’ willingness to take action was influenced by their attitudes, behavioural control and trust in wildlife/land managers, whereas socio-demographic characteristics were less influential. These findings help us to understand visitor behaviour with respect to amphibian biosecurity in natural areas and inform enhanced biosecurity measures and strategic messaging to reduce pathogen spillover.