Background: The sand fy Nyssomyia neivai is one of the most abundant species in Southern Brazil. It is frequently found in areas that are foci of visceral leishmaniasis in the state of Santa Catarina, caused by Leishmania infantum. In this region, the main vector of L. infantum, Lutzomyia longipalpis, has not been detected. In the absence of L. longipalpis, this study aimed to identify the sand fy fauna and diagnose any potential Leishmania spp. infection in sand fies and in dogs in a region of Southern Brazil that experienced a recent canine visceral leishmaniasis outbreak. Methods: This report includes a survey of the sand fy fauna at the Zoonosis Control Center of the Municipality of Tubarão (Santa Catarina, Brazil). Molecular tests were conducted to investigate Leishmania spp. natural infection in sand fies using polymerase chain reaction (PCR). In positive females, in addition to morphological identifcation, molecular analysis through DNA barcoding was performed to determine the sand fy species. Additionally, the dogs were tested for the presence of Leishmania spp. using a non-invasive technique for the collection of biological material, to be assessed by PCR. Results: A total of 3419 sand fies, belonging to fve genera, were collected. Nyssomyia neivai was the most abundant species (85.8%), followed by Migonemyia migonei (13.3%), Pintomyia fscheri (0.8%), Evandromyia edwardsi (<0.1%), and species of the genus Brumptomyia. (0.1%). Out of the 509 non-engorged females analyzed by PCR, two (0.4%) carried L. infantum DNA. The naturally infected females were identifed as Ny. neivai, in both morphological and molecular analysis. In addition, two out of 47 conjunctival swabs from dogs tested positive for L. infantum, yielding an infection rate of 4.2%. Conclusions: These results confrm the presence of Ny. neivai naturally infected with L. infantum in an area where dogs were also infected by the parasite, suggesting its potential role as a vector in Southern Brazil.