The growing international trade and global seafood consumption rate have heightened the risks of fisheries crime, economic fraud, and the endangerment of marine wildlife. In this industry, the introduction of more species into trade makes authentication and species identification challenging for consumers and even taxonomists also due to morphological similarities (e.g., cryptic species) and the complexity of seafood processing, such as mincing or mixing (e.g., shark meat and turtle egg soup). These challenges are intensified with the proliferation of raw and processed items where morphological distinctions are lost. This complexity hampers efforts to verify product authenticity and prevent seafood mislabeling fraud. While molecular biology has been widely adopted for species confirmation, large-scale seafood authentication in markets and restaurants could be challenging due to high costs and the time required for testing a limited number of samples. Environmental DNA (eDNA) metabarcoding is a revolutionary technique for seafood authentication (even highly processed seafood) and the conservation of endangered species. This chapter discusses the overview of eDNA metabarcoding, detailing its principles and applications. We explore the potential of this robust, cost-effective molecular tool to combat seafood fraud, illegal fishing, and accurate traceability throughout the supply chain. Furthermore, the chapter highlights the technology’s capacity to enhance transparency by offering consumers and regulatory bodies unprecedented access to information about the origin and composition of seafood products.