West Nile virus (WNV) and Usutu virus (USUV) are zoonotic arboviruses. These flaviviruses are mainly maintained in the environment through an enzootic cycle involving mosquitoes and birds. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Nevertheless, there is little data regarding the involvement of other mammals in the epidemiology of these arboviruses. In this study, we performed a serosurvey to assess exposure to these viruses in captive birds and mammals in a zoo situated in the south of France, an area described for the circulation of these two viruses. A total of 411 samples comprising of 70 species were collected over 16 years from 2003 to 2019. The samples were first tested by a competitive enzyme-linked immunosorbent assay. The positive sera were then tested using virus-specific microneutralization tests against USUV and WNV. USUV seroprevalence in birds was 10 times higher than that of WNV (14.59% versus 1.46%, respectively). Among birds, greater rhea (Rhea Americana) and common peafowl (Pavo cristatus) exhibited the highest USUV seroprevalence. Infections occurred mainly between 2016–2018 corresponding to a period of high circulation of these viruses in Europe. In mammalian species, antibodies against WNV were detected in one dama gazelle (Nanger dama) whereas serological evidence of USUV infection was observed in several Canidae, especially in African wild dogs (Lycaon pictus). Our study helps to better understand the exposure of captive species to WNV and USUV and to identify potential host species to include in surveillance programs in zoos.