Understanding the processes that affect freshwater ecosystems at the watershed level is fundamental for the conservation and management of river otters. During 2 consecutive years, we surveyed the occurrence of the Southern river otter Lontra provocax and its main prey (crustaceans) in a watershed of 9900 km2 in the Chilean temperate forest. We modeled predator and prey distributions with a variety of statistical techniques by relating a set of environmental predictors to species occurrence records. Otter and crustaceans were associated with areas of intermediate to low human disturbance with a mosaic of riparian vegetation densities, mainly at low altitudes. The singularity of the Andean Range, with a very marked elevation gradient and oligotrophic watercourses in the higher areas, created more vulnerable conditions for otter presence because prey abundances were limited in those areas. Human impacts affected otter populations at a landscape scale through the presence of main roads, as these were mostly located in lower parts of the watershed where otters have their primary habitat. These results point to the importance of land management and protection of low-elevation areas where otters still occur to ensure the long-term viability of its freshwater populations.