A serological survey was performed to evaluate the presence of specific antibodies against Neospora caninum in dogs and native animals in a wildlife conservation area in southern Portugal. The study involved 463 animals, including dogs (n = 286), European rabbits (Oryctolagus cuniculus; n = 32), Egyptian mongoose (Herpestes ichneumon; n = 34), wild boars (Sus scrofa; n = 26), foxes (Vulpes vulpes, n = 25), common genets (Genetta genetta; n = 17), red deer (Cervus elaphus; n = 14), wildcats (Felis silvestris; n = 6), four mustelid species (n = 17) and rodents (n = 6). Samples from dogs were analysed by Indirect Fluorescent Antibody Test (IFAT). Samples from wild animals were screened by the modified agglutination test and positive and doubtful results were confirmed by IFAT. The seroprevalence of N. caninum in dogs was 32·5% [95% confidence interval (95% CI) 27·3–38·1]. Among wild animals, exposure to N. caninum was confirmed only in foxes (12%, 95% CI 4·2–30) and rabbits (25%, 95% CI 13·3–42·1). This is the first evidence of natural exposure to N. caninum in foxes and rabbits in Portugal, and our results suggest that rabbits may play a role as reservoirs of infection to dogs, foxes and other wildlife carnivores. The relevance of this finding in the sylvatic cycle of N. caninum needs further studies, since infection may affect wildlife species and cattle grazing in the same areas.