The ongoing expansion of global trade and human mobility are causing increased occurrences of biosecurity outbreaks, particularly of invasive pests which threaten biodiversity and agricultural production. Smallholder farmers are particularly vulnerable, as they are generally not well-prepared for such incursions. In Australia, farmers with non-English speaking backgrounds are more likely to be influenced by these increasing risks and may need long-term adaptive support. Understanding farmers’ perceptions of biosecurity threats, and what drives these perceptions, is a prerequisite for designing effective biosecurity management policies to support livelihoods and food security. In this study, we aim to understand the perceptions of smallholder farmers’ on biosecurity threats and the factors influencing their perceptions. Data was obtained from a survey of 101 Vietnamese farmers across three geographically dispersed locations in Australia (Northern Territory, South Australia, and Western Australia). Using the Protection Motivation Theory, we found that 43% of farmers perceived biosecurity threats to be one of the key agricultural risks, preceded by weather-related risk (48%) and produce price fluctuations (45%). Belief in the existence of biosecurity threats and the sources of information on biosecurity matters had a significant positive influence on farmers’ overall perceptions of biosecurity threats on four dimensions of their life. English levels had a significant influence on their perceptions of biosecurity threats on crop output whereas farm size, gender, and farmers’ age did not significantly affect farmers’ overall perceptions of biosecurity threats. The findings of this study could be used by government agencies and industry associations to better target support for farmers to minimise the threats of pest and disease incursions. Support could be delivered through the introduction of relevant extension services and education programs with more transparent and direct communications of risk and compliances, improving farmers’ knowledge and belief in biosecurity and fostering trust between farmers and the government.