This dataset contains the eDNA data collected as a part of the UNESCO eDNA expeditions in Marine World Heritage Sites project in 2022-2024. The project was coordinated in collaboration with the Ocean Biodiversity Information System (OBIS) of IOC-UNESCO and the marine programme of the UNESCO World Heritage Center, and funded by the government of Flanders (Kingdom of Belgium). The samples were collected with citizen science eDNA sampling kits, containing a syringe and a filter, and preserved with Longmire's buffer, before sequencing at a central laboratory. Five different biomarkers were analysed, including 12SMifish, Mimammal, Teleo, 16S-Vert and COI markers. The sequences were analysed with the PacMAN pipeline and quality controlled including a validation check by local scientists and site managers as well as compared to existing knowledge on species distributions in OBIS and GBIF. The full sampling protocols and materials are available on the project website (https://www.unesco.org/en/edna-training-materials?hub=66910), and the bioinformatics pipeline is on github (https://github.com/iobis/PacMAN-pipeline). The data can also be explored on the eDNA expeditions dashboard (https://dashboard.ednaexpeditions.org/).