Protecting plants from new pathogen incursions requires effective surveillance practices. Environmental DNA (eDNA) metabarcoding shows considerable promise for detecting invasive organisms in terrestrial and aquatic ecosystems. Metabarcoding is widely used to characterise plant microbiotas and is beginning to be applied to plant biosecurity. However, diagnostic assays for biosecurity must fit within internationally agreed frameworks. Development of new gene targets to improve taxonomic resolution, improved reference databases and simplified bioinformatics platforms are required before phytopathogenic bacteria and fungi can be routinely detected by metabarcoding. Building biodiversity maps from accumulated metabarcoding data represents an important opportunity to define organism presence/absence in New Zealand and Australia and thus to enhance biosecurity decision making. Advances in sequencing technologies and infrastructure promise the creation of eDNA biosecurity surveillance networks from substrates including soil, trapped spores and insects.