The global ornamental fish trade enables translocation of exotic aquatic pathogens. In many countries, health certification and visual inspection of imported fish are key components of biosecurity to prevent the introduction of aquatic diseases. However, infected fish do not always exhibit clinical or behavioural signs of disease, and alternatives to visual inspection must be validated. This study examined the use of environmental DNA (eDNA) to detect sub-clinical parasite infections at border control. We simulated the export process of live ornamental fish in which non-infected fish, infected fish, treated fish, and non-infected fish held in contaminated water were packaged and delivered in 48 h. Quantitative PCR (qPCR) was used to detect eDNA of an ectoparasitic monogenean, Neobenedenia girellae, infecting barramundi, Lates calcarifer. The qPCR assay did not reliably detect parasite eDNA under 2 copies/µL from fish with sub-clinical infections (mean parasite intensity = 6.80 ± 4.78 S.D.), suggesting parasite infection loads tested in this study may be too low for reliable detection within the timeframe used to export live ornamental fish. Quantitative PCR tests detected parasite eDNA in 50% of infected fish and 70% of non-infected fish in contaminated transport water. This indicated a high plausibility of false negative detections because of low eDNA concentrations in transport water and false positive detections of DNA from dead parasites in the water. Environmental DNA screening methods for border control biosecurity must overcome limitations posed by low eDNA concentrations in the water, limited timeframes for sample processing, and the essential differentiation between live parasite infections and dead, non-viable parasites.