AbstractThis special issue focuses on the applications of environmental DNA (eDNA) sequencing for the detection and monitoring of disease‐causing agents, including viruses, bacteria, protozoans, myxozoans, fungi, trematodes, and arthropods. We explore the impact of eDNA technologies, such as metabarcoding and qPCR, in understanding the dynamics of pathogens in various environments as well as their implications for conservation, biosecurity, and veterinary and agricultural health under the “One Health” framework. This issue addresses how molecular sequencing provides innovative solutions to the challenges faced by conventional parasite and pathogen detection methods, enabling a more comprehensive understanding of the spatiotemporal dynamics of disease agents. Finally, we discuss the challenges in eDNA applications, such as primer development and taxonomic resolution, and the opportunities for future research in advancing eDNA methodologies for infectious disease studies. This issue highlights the growing importance of eDNA surveillance in understanding and managing the health of ecosystems and at‐risk species.