Ilulissat Icefjord in Greenland is experiencing the effects of climate change, with the Sermeq Kujalleq glacier being one of the fastest-moving and most productive ice streams in Greenland. This is likely affecting the distribution of species in the fjord, including those important to local fisheries. Due to heavy ice conditions, few studies on environmental and ecological conditions exist from the fjord. However, new techniques such as environmental DNA (eDNA) metabarcoding now allow deeper insight into the fjord system. Here, we combine local ecological knowledge with data on hydrographic conditions, stable isotopes (delta 18O), and eDNA metabarcoding to investigate the spatial and seasonal distribution of marine fish and mammals inside Ilulissat Icefjord. Our eDNA results support local observations that Arctic char migrate to the southern fjord during summer, harp seals forage in large herds in the fjord system, polar cod is the dominant prey fish in the area, and Greenland shark likely does not reside in the fjord system. Lower predation pressure in the Icefjord, due to the absence of Greenland shark and polar bears as well as limited fishing/hunting, is presumably one of the reasons why ringed seals and Greenland halibut are larger in the Icefjord. Furthermore, our results indicate that in summer, the southern branch of the fjord system has a more diverse community of vertebrates and different water masses than the northern branch and main fjord, indicating a time lag between inflows to the different branches of the fjord system. Our approach highlights the value of combining local ecological knowledge with scientific research and represents a potential starting point for monitoring biological responses in Ilulissat Icefjord associated with climate-induced changes.