Understanding human influence on ecosystems and their services is crucial to achieve sustainable development and ensure the conservation of biodiversity. In this context, the human footprint index (HFI) represents the anthropogenic impacts on ecosystems and the natural environment. Our objective was to characterize the HFI in Southern Patagonia (Argentina) across the landscape, qualifying the differences among the main ecological areas and especially the forested landscapes. We also assessed the potential utility of HFI to identify priority conservation areas according to their wilderness quality and potential biodiversity values. We created a HFI map (scores varied from 0 representing high wilderness quality to 1 representing maximum human impact) using variables related to direct (e.g. infrastructure) and indirect (e.g. derived from economic activities) human impacts, including settlements, accessibility, oil industry, and sheep production. HFI varied significantly across the natural landscapes, being lower (0.07−0.11) in remote ecosystems close to the Andes Mountains and higher (0.38−0.40) in southern areas close to the provincial capital city. Forested landscapes presented different impact values, which were directly related to the economical values of the different forest types. We determined that the current protected area network is not equally distributed across the different ecological areas and forest types. Priority conservation areas were also identified using the fragmentation produced by the human impact, the patch size, and the potential biodiversity values. HFI can present high compatibility with other land-use management decision making tools, acting as a complement to the existing tools for conservation planning or management.