Aquila chrysaetos and Aquila fasciata are two congeneric eagle species distributed in the Mediterranean region which are supposed to compete for similar breeding and foraging resources. In the present study, bioclimatic, topographic, and human-related habitat parameters were investigated for 64 and 75 nest sites of Golden and Bonelli’s eagles, respectively. The nests were found during fieldwork undertaken from 1995–2020. Overall, the habitat parameters that best discriminated nest site selection were associated with elevation, temperature, and land use with topographic variables being most powerful for niche separation. Univariate analysis, regression, and species distribution modeling identified a strong association of the species with altitude pinpointing the Golden eagle’s mountainous and continental character and the Bonelli’s eagle being a lowland and coastal species. Golden eagle nests were situated away from human settlements on steep cliffs in higher altitude areas with transitional woodland-shrub vegetation. In contrast Bonelli’s eagle nests were located on low-altitude warmer zones, closer to the coast and human settlements with more natural grasslands in their vicinity. The ecological niche separation of the two species was best described by altitude and temperature, though no clear-cut evidence was detected for their competitive exclusion. Inter-specific nearest neighboring distance was found statistically significant only for the Golden eagle which seems to be less tolerant in its co-existence with the Bonelli’s eagle. Conservation measures for both species should target territories under human pressure, though more research should focus on the species range use and habitat heterogeneity within overlapping territories.