The increase in human population and domestic pets, such as cats, are generating important consequences in terms of habitat loss and pathogen pollution of coastal ecosystems with potential to generate negative impacts in marine biodiversity. Toxoplasma gondii is the etiological agent of zoonotic disease toxoplasmosis, and is associated with cat abundance and anthropogenic disturbance. The presence of T. gondii oocysts in the ocean has negatively affected the health status of the threatened Southern sea otter (Enhydra lutris nereis) populations. The present study analyzed seroprevalence and presence of T. gondii DNA in American mink (Neovison vison), Southern river otters (Lontra provocax) and domestic cats (Felis silvestris catus) in four different areas in Southern Chile comprising studies in rivers and lakes in Andean foothills and mountains, marine habitat and island coastal ecosystems. Mean seroprevalence of T. gondii in the study was 64% of 151 total animals sampled: 59% of 73 American mink, 77% of 13 Southern river otters, 68% of 65 domestic cats and in two of two kodkods (Leopardus guigna). Toxoplasma gondii DNA was detected in tissues from one American mink and one Southern river otter. The present study confirms the widespread distribution of T. gondii in Southern Chile, and shows a high exposure of semiaquatic mustelids and domestic cats to the parasite. Cats and anthropogenic disturbance have a role in the maintenance of T. gondii infection in ecosystems of southern Chile.