AbstractMesophotic marine ecosystems are characterized by lower light penetration supporting specialized fish fauna. Due to their depths (−30–−150 m), accessibility is challenging, and the structure of mesophotic fish assemblages is generally less known than either shallow reefs or deep zones with soft bottoms which are generally trawled. Environmental DNA metabarcoding from seawater filtered in situ could improve our ability to monitor the diversity of mesophotic ecosystems. Here, we developed and tested a submersible standalone pumping device allowing targeted marine water filtering to explore the biodiversity of two mesophotic ecosystems, one temperate along the Provence coast in the North‐Western Mediterranean Sea and one tropical at the seamount La Pérouse in the Western Indian Ocean. We filtered water samples from depths ranging between 0 and 200 m in the Mediterranean Sea and between 60 and 140 m in the Indian Ocean and applied a metabarcoding protocol using the teleo primer pair targeting the 12S mitochondrial rDNA (Actinopterygii and Chondrichthyes). For both study regions, our eDNA surveys were able to recover highly diverse fish assemblages, and the compositional analysis of eDNA samples showed both a marked signal of fish compositional turnover and overlapping taxa between depth zones. Further, we observed that a substantial number of species were found in samples collected in depths beyond their reported depth range suggesting an underestimation of species' depth tolerances. eDNA metabarcoding should thus complement existing knowledge of species' geographic distributions across space and depth. Overall, our results demonstrate the potential of eDNA metabarcoding for future mesophotic surveys as it allows fast and broad biodiversity assessment.