Vultures provide crucial ecological, economic, and cultural benefits, yet many Old World populations are declining. The illegal trade in vultures for traditional medicines (known as muthi in South Africa) is widely regarded as an important threat to the conservation of African vultures, but there are relatively few studies on the acquisition and trade in vultures for traditional medicine, which limits our understanding (and the effective mitigation) of this threat. We assessed the use of vultures in traditional medicine in the Kruger to Canyons Biosphere Region, South Africa, by conducting questionnaires with 51 traditional healers from the Kukula Traditional Health Practitioners Association in Bushbuckridge, Mpumalanga Province. Traditional healers reported that vulture body parts were used alone, or in combination with plants or minerals, for various purposes, primarily to give clairvoyant abilities, but also to promote good dreams or increased intelligence, to treat illnesses, and to appease the ancestors. Vultures were acquired (for use in traditional medicine) by poisoning and trapping in communal rangelands and inside protected areas (the Kruger National Park, Manyeleti Game Reserve, Sabi Sands Game Reserve, and Bushbuckridge Nature Reserve). Vultures reportedly varied in price from ZAR300 to ZAR1500 (approximately US$17 to US$85) for a whole bird. We estimate that this association of traditional health practitioners uses 400–800 vultures a year. Our survey respondents expressed concern that acquiring vultures through poisoning is unsustainable, because birds are killed en masse, and this may reduce the sustainability of vulture populations for traditional use by future generations. Our respondents suggested the Greater Honeyguide (Indicator indicator) and honey badger (Mellivora capensis) as alternatives for vultures in traditional medicine. There is a need for better law enforcement in our study area to reduce the threat that illegal killing for traditional medicine poses to the region's vultures.