Many emerging infectious diseases are zoonotic and transmitted by an arthropod vector; thus, the Global One Health perspective is necessary for a better understanding of these diseases. Bartonellosis, one of those emerging infectious diseases, is caused by bacteria of the genus Bartonella, and can have different clinical manifestations in humans and animals. Opossums and their ectoparasites have been linked to human-wildlife conflicts. Knowing this, we aimed to evaluate infection by Bartonella spp. in free-living opossums seized or rescued from Atlantic Forest biome of Rio de Janeiro (southeastern Brazil). Whole blood was obtained from seven Didelphis aurita and eight D. albiventris, and DNA was assessed by conventional and quantitative polymerase chain reactions (PCR and qPCR). Positivity was achieved in 40.0% (PCR) and 46.7% (qPCR) of the samples, revealing, possibly for the first time, the presence of Bartonella spp. DNA in opossums from peri-urban areas of Atlantic Forest biome. Studies should continue, in order to elucidate synanthropic mammals importance in bartonellosis epidemiology.