The marine ecosystem of Kongsfjorden (KF), Svalbard, is directly affected by ongoing climate change in the Arctic. Shifts in species composition and distributions are already underway as a result of the transition from Arctic to Atlantic conditions in the fjord. The polar night is a period of challenging conditions and is historically understudied, and thus our understanding of the biodiversity of major eukaryotic groups is particularly limited at this time of year. Here we aimed to provide a comprehensive snapshot of eukaryotic biodiversity present in KF during the polar night using environmental DNA (eDNA) metabarcoding of the mitochondrial cytochrome c oxidase subunit 1 gene (COI). Additionally, we sought to establish a baseline dataset for gelatinous zooplankton (GZP) during this period by combining eDNA with net sampling. Lastly, we tested for the impact of PCR-inhibition on the recovery of eDNA from turbid fjord waters. We successfully generated species lists for a large component of the pelagic community known to inhabit KF, benthic and hyperbenthic species typical to the area, as well as a number of new detections. We recovered taxa from major functional groups in the fjord, including macroalgae, phytoplankton, zooplankton, large vertebrates and benthic invertebrates. Finally, we recovered a richer polar night GZP community than previously detected with morphology-based methods, including other seasons. This study demonstrates the versatility of eDNA metabarcoding for marine biodiversity surveys in challenging environmental conditions and provides a baseline for future polar night eDNA-based biodiversity monitoring in KF.