Effective border control relies on stringent biosecurity protocols to detect and prevent introductions of exotic pests and diseases. Detection of pathogens and parasites in the live ornamental fish trade using environmental DNA (eDNA) techniques has the potential to improve current biosecurity practices. We examined water samples from 11 target consignments (cyprinids susceptible to Dactylogyrus spp. infections) and seven non-target fish consignments (non-cyprinids, not susceptible to Dactylogyrus spp. infections) imported from Southeast Asia to Australia for the presence of eDNA from five Dactylogyrus species (Monogenea: Dactylogyridae). A four-step predictive framework was used to predict putative positive and putative negative detections from quantitative PCR assays. Both target and non-target consignments were positive for Dactylogyrus spp. eDNA as confirmed by Sanger sequencing. Positive detections for Dactylogyrus spp. eDNA in non-target fish consignments demonstrates the possibility of source water contamination, limiting the applicability of eDNA screening methods at border control. This study suggests that screening for parasite eDNA within ornamental fish consignments should be tested during pre-export quarantine periods to avoid false positive detections at border control. Lastly, the proposed predictive framework has a broad utility for minimizing false positive and false negative eDNA detections of aquatic organisms.