Tis study evaluated the presence of Neospora caninum and Toxoplasma gondii antibodies in 320 domestic dogs living in urban and rural areas of the municipality of Poconé, located in the Pantanal wetlands of the state of Mato Grosso, Brazil. Anti-N. caninum (cut-of point 50) and anti-T. gondii (cut-of point 16) antibodies were detected by means of immunofuorescence assays (IFA), using NC-Bahia and RH strains as antigens, respectively. Anti-N. caninum antibodies were detected in 69 (21.56%; 95% CI: 17.27%-26.56%) dogs, 31 (44.93%) of which lived in urban areas and 38 (55.07%) in rural areas, and endpoint titers ranged from 50 to 3200. Anti-T. gondii antibodies were found in 132 (41.25%; 95% CI: 35.84%-46.87%) dogs, 58 (43.94%) from urban areas and 74 (56.06%) from rural areas, and endpoint titers ranged from 16 to 8192. A total of 33 dogs (10.3%) (12 urban and 21 rural animals) reacted to both agents (P <0.05). Tis study showed a higher prevalence of anti-T. gondii compared to anti-N. caninum antibodies in the dogs living in the Pantanal region, suggesting that both protozoans circulate in the studied region. Tis information is relevant, in view of its implications for animal and public health.