Mesophotic coral ecosystems (MCEs), particularly those in remote locations such as seamounts and banks, remain among the least explored reef habitats due to limited accessibility. Nevertheless, these ecosystems harbour high diversity and understanding the taxonomic and functional composition of their communities has become increasingly important under growing anthropogenic pressures. Environmental DNA (eDNA) metabarcoding provides a powerful approach for characterizing biodiversity in these remote reefs, where conventional surveys are limited by accessibility and bottom-time constraints. In this study, we applied a multi-marker eDNA metabarcoding approach (CO1, ITS2, 12S) to seawater and sediment samples to compare the diversity of benthic and fish communities on shallow (4–10 m) and upper mesophotic (43–50 m) coral reefs of two Caribbean offshore banks, and two nearby islands in the Cayman Islands. We found that benthic and fish community compositions varied significantly (1) along the depth gradient from shallow to upper mesophotic coral reefs as well as (2) between the two offshore banks and two island reef systems studied. While some typical coral reef fish such as grunts and parrotfishes were significantly more abundant on the islands’ mesophotic reefs, the banks’ unique community composition was partially driven by higher relative abundance of pelagic and deep-sea species, suggesting higher productivity and a potential function as aggregation points for fishes. A comparatively high relative abundance of several reef-building scleractinian corals on the offshore banks, together with the detection of overexploited and vulnerable fish species, highlights the importance of extending conservation measures already implemented on the islands to these offshore ecosystems. These results demonstrate the potential of multi-marker eDNA metabarcoding to characterize biodiversity patterns across depth and among reef formations, highlighting its value for monitoring remote reef ecosystems that remain underrepresented in biodiversity assessments and conservation planning.