The effects of climate change, including warming waters, loss of sea ice habitat and the resulting changes in primary production, are inducing shifts in marine communities across the Arctic Ocean. The Arctic marginal ice zone (MIZ) is a highly dynamic ecosystem and is a transition zone between pack ice and the open ocean. It is habitat for a wide range of organisms, including sympagic and pelagic taxa, all of which are affected by the changing physical dynamics of the MIZ. Here we use a multi-marker (18S rRNA V1-2 and COI Leray-XT) approach to investigate eukaryotic biodiversity of the upper water column in this understudied habitat. Environmental DNA (eDNA) was sequenced from seawater samples collected directly beneath the sea ice and at a depth of 5 m, sourced from ice floes representing different ice regimes. To explore the abiotic factors influencing under-ice diversity, we combined satellite-derived environmental data with simultaneous in situ hydrographic measurements. Our analysis identified a range of sympagic and pelagic metazoans, along with primary producers typical of the region, as well as substantial uncharacterised diversity. Alpha diversity indices were higher immediately below the ice, and community composition differed across depths and ice floe stations. We show that the properties of the meltwater stratification in the upper ocean, along with sea ice concentration and distance to the ice edge, significantly shape eukaryotic diversity and community composition. These findings highlight the effectiveness of eDNA metabarcoding for monitoring sub-ice communities and enhance our understanding of eukaryotic biodiversity in the MIZ.