Biodiversity and economic losses resulting from invasive plant pests and pathogens are increasing globally. For these impacts and threats to be managed effectively, appropriate methods of surveillance, detection and identification are required. Botanical gardens provide a unique opportunity for biosecurity as they accommodate diverse collections of exotic and native plant species. These gardens are also often located close to high-risk sites of accidental invasions such as ports and urban areas. This, coupled with routine activities such as the movement of plants and plant material, and visits by millions of people each year, place botanical gardens at risk to the arrival and establishment of pests and pathogens. Consequently, botanical gardens can pose substantial biosecurity risks to the environment, by acting as bridgeheads for pest and pathogen invasions. Here we review the role of botanical gardens in biosecurity on a global scale. The role of botanical gardens has changed over time. Initially, they were established as physic gardens (gardens with medicinal plants), and their links with academic institutions led to their crucial role in the accumulation and dissemination of botanical knowledge. During the second half of the 20th century, botanical gardens developed a strong focus on plant conservation, and in recent years there has been a growing acknowledgement of their value in biosecurity research as sentinel sites to identify pest and pathogen risks (novel pest-host associations); for early detection and eradication of pests and pathogens; and for host range studies. We identify eight specific biosecurity hazards associated with botanical gardens and note potential management interventions and the opportunities these provide for improving biosecurity. We highlight the value of botanical gardens for biosecurity and plant health research in general, and the need for strategic thinking, resources, and capacity development to make them models for best practices in plant health.