The application of environmental DNA (eDNA) and RNA (eRNA) technologies to aquatic ecosystem monitoring and management has increased rapidly in the last decade. These methods are providing many new and exciting opportunities for enhanced biodiversity assessment, ecological health evaluation, and species detection. This special issue of PeerJ Life and Environment brings together 20 innovative studies that collectively advance the eDNA toolkit. Four key themes are covered: (i) Methodological advancements, (ii) Ecological health assessments and biomonitoring, (iii) Species detection, and (iv) Application and management. The studies cover a suite of topics including; optimizing sample collection, developing species-specific assays, evaluating bioindicator species, assessing microbial activity, and biodiversity monitoring in diverse freshwater and marine habitats. Emerging applications, such as the use of genome skimming to identify new fish markers, showcase the many new advancements in this field. The studies in this issue also highlight challenges, including the need for standardized protocols and ethical considerations that must be addressed before these tools can be implemented or adopted for decision making at national or global scales. Together, these contributions demonstrate the transformative potential of environmental nucleic acids’ technologies for advancing aquatic conservation and management. By bridging methodological rigor with applied research, the studies in this special issue provide an important resource for researchers, policymakers, and practitioners committed to sustainable aquatic ecosystem stewardship.