In 1997, Eurasian Eagle Owls Bubo bubo returned to Limburg, the south-eastern province of The Netherlands. However, since then the population size has stagnated, while extremely high concentrations of PCBs have been found in Eagle Owl carcasses. These observations suggest that a further expansion of the species in Limburg is under threat. The high concentrations of toxic substances may be explained by the Eagle Owls' position at the top of the food chain. Studying their hunting behaviour may help to identify possible sources of poisoning. Identifying the most important habitat characteristics of hunting grounds is a first step towards recognizing poisoning sources. For this purpose, and to gain general insight into their habitat use, a GPS tracking study was conducted on six adult Eagle Owls from three adjoining territories in 2010–2011. We found that the quarries in the region are important for the Eagle Owls since they are used as nesting sites. Other habitat characteristics that attract the species are pastures, differences in altitude, forest edges and water bodies. Major roads and town centres are avoided. The highlighted areas and habitat characteristics provide information about the most important hunting habitats of Eagle Owls and insight into the underlying causes threatening this species in Limburg.