West Nile virus (WNV) is pathogenic in a wide range of avian hosts and is endemic in much of North America. This virus is responsible for population declines of some Passeriformes. We describe a WNV-associated mortality event in American White Ibis (Eudocimus albus) nestlings. This is a species, inherent to the Everglades ecosystem, which has recently begun nesting in urban areas. An urban colony in south Florida was monitored from March–July in 2020 as part of an ongoing study. Nestling carcasses were collected opportunistically and sent to the Southeastern Cooperative Wildlife Disease Study, University of Georgia within 24 h for diagnosis. Three ibis nestling deaths were confirmed to be caused by WNV infection based on histopathology, immunohistochemistry, and reverse transcription PCR. Serial plasma samples collected weekly from 36 healthy chicks of the same urban rookery were tested for WNV-neutralizing antibodies via plaque reduction neutralization test; four chicks were seropositive. Antibody titers in three seropositive chicks from which serial samples were collected waned over time, suggesting maternal antibody transfer. Ibis mortalities were consistent with a spike of WNV activity in this region of Florida. West Nile virus infection may be an important seasonal cause of mortality for wading bird nestlings.