The aims of this study were to investigate the presence of Leishmania infantum and possible co-infection with Anaplasma platys, Babesia vogeli, Ehrlichia canis, and Toxoplasma gondii in the brain of 24 dogs naturally infected by L. infantum. A total of 24 mongrel adult dogs (22 clinically affected, 2 with neurological signs, and 2 subclinically infected) aged between 2 and 5 yr, naturally infected by visceral leishmaniasis, were selected. Fragments from meninges, frontal cortex, thalamus, cerebellum, and choroid plexus of the lateral ventricles and fourth ventricle were collected, mixed, and tested by real-time polymerase chain reaction (qPCR). Leishmania infantum DNA was detected in 95.8% (23/24) of the infected dogs, including the subclinically infected. A total of 14/24 (58.3%) dogs were co-infected by E. canis and L. infantum, 4/24 (16.7%) were co-infected by E. canis, B. vogeli, and L. infantum, 2/24 (8.3%) were co-infected by B. vogeli and L. infantum, and 1/24 (4.2%) dog was co-infected by E. canis, B. vogeli, T. gondii, and L. infantum. All 24 brain samples tested negative for A. platys. These results demonstrate that L. infantum is able to penetrate into the brain parenchyma, either alone or in association to other zoonotic pathogens. In addition, qPCR could be considered for adequate evaluation of Leishmania in the brain tissue of dogs with neurological signs that have died.