The Arctic Ocean is home to a unique fauna that is disproportionately affected by global warming but that remains under-studied. Due to their high mobility and responsiveness to global warming, cephalopods and fishes are good indicators of the reshuffling of Arctic communities. Here, we established a nekton biodiversity baseline for the Fram Strait, the only deep connection between the North Atlantic and Arctic Ocean. Using universal primers for fishes (12S) and cephalopods (18S), we amplified environmental DNA (eDNA) from seawater (50–2700 m) and deep-sea sediment samples collected at the LTER HAUSGARTEN observatory. We detected 12 cephalopod and 31 fish taxa in the seawater and seven cephalopod and 28 fish taxa in the sediment, including the elusive Greenland shark (Somniosus microcephalus). Our data suggest three fish (Mallotus villosus, Thunnus sp., and Micromesistius poutassou) and one squid (Histioteuthis sp.) range expansions. The detection of eDNA of pelagic origin in the sediment also suggests that M. villosus, Arctozenus risso, and M. poutassou as well as gonatid squids are potential contributors to the carbon flux. Continuous nekton monitoring is needed to understand the ecosystem impacts of rapid warming in the Arctic and eDNA proves to be a suitable tool for this endeavor.