The comparability of methods applied to environmental DNA (eDNA) samples across laboratories remains a significant challenge for international biodiversity monitoring projects. Inconsistently performing practices can jeopardize the reliability of data that is essential for effective conservation strategies across geographic regions and focal species. To address potential discrepancies among four international partner laboratories who are part of a collaborative eDNA initiative, a ring test was conducted to compare extraction efficiencies based on 39 eDNA samples. Each laboratory contributed eight to eleven eDNA samples collected from five locations throughout the North-East Atlantic and Mediterranean Sea near marine megafauna (whales, dolphins, and sharks). After lysis, aliquots were exchanged between laboratories then independently extracted using each facility’s preferred method. Extracts were then returned to their respective laboratories of origin for measurements of total DNA concentration as well as quantitative PCRs using species-specific assays designed for each associated target species. Our findings revealed similar concentrations of total DNA, yet a significant deviation in extraction performance for targeted qPCR reactions by one laboratory. Overall, detection success differed based on the target taxa with sharks being less often detected (and at lower concentrations) than whales and dolphins. Significant interaction effects were found between combinations of laboratories and species, suggesting that particular extraction protocols may be most efficient for specific environmental conditions. Our study serves as a foundational step towards establishing rigorous, reproducible eDNA practices that are crucial for the success of multinational environmental monitoring projects to enable the direct comparison of results.