A near-threatened species, the maned wolf’s ( Chrysocyon brachyurus ) range extends from central Brazil, northern and eastern Bolivia, and southeastern Peru to the southeast of the Gran Chaco ecoregion and adjacent scrubland in Argentina. Rural domestic dogs under ownership may pose threats to maned wolves as reservoir hosts of multiple pathogens. A serologic survey of rural domestic dogs and maned wolves was conducted to document exposure to canine pathogens in southeastern Santiago del Estero, Argentina, with a parallel questionnaire of knowledge, attitudes, and perception of local villagers in relation to maned wolves. The three maned wolves examined had evidence of exposure to canine adenovirus, canine distemper virus, canine parvovirus, Leptospira interrogans spp., and Dirofilaria immitis . Dogs had very high seroprevalence for Neospora caninum and canine coronavirus (67%), canine adenovirus (59%), D. immitis (58%), canine distemper virus (57%), Toxoplasma gondii (33%), and L. interrogans spp. (20%). Antibodies for canine parvovirus, Brucella canis and Trypanosoma cruzi were rare or absent. Multiple logistic regression analysis showed that dog seroprevalence was not significantly associated with age, sex, and function for any of the pathogens investigated. The frequency distribution of seroreactivity to specific pathogens per dog was highly aggregated. Villagers considered that illegal trade, hunting, vehicular collisions, and interactions with dogs were the most serious local threats for maned wolves. The multiple threats faced by the maned wolf populations in southeastern Santiago del Estero suggest that their populations are unlikely to remain viable unless specific protection measures are taken.