Using a suite of trapping techniques, biting flies were collected at the Riverbanks Zoo (Columbia, SC) and the Greenville Zoo (Greenville, SC) for three consecutive years. The most frequently collected biting flies were mosquitoes (Culicidae) followed by biting midges (Ceratopogonidae), biting muscids (Muscidae), black flies (Simuliidae), and deer flies (Tabanidae). Flies were tested for the presence of several arboviruses, bacterial pathogens, and protozoan pathogens. Blood meals were identified in blood-fed flies using molecular assays with a vertebrate-specific primer pair targeting a portion of the cytochrome b gene. We found that Aedes albopictus (Skuse) feeds on wallabies, gorillas, orangutans, several monkey species, humans, and horses. The biting midge Culicoides guttipennis (Coquillett) was associated with a colobus monkey and a golden-headed lion tamarin. Aspects of biting fly host specificity, phenology, and vector management within zoological gardens are discussed.