Several rabies-related lyssaviruses have been associated with bat species in southern Africa, the rarest of these being Duvenhage virus (DUVV), for which only five isolations have been made over five decades. Three of these were from human fatalities, and it is not known which bat species acts as reservoir. In studying a population of Nycteris thebaica in the kingdom of Swaziland, a landlocked country bordering Mozambique and South Africa, we found evidence of the circulation of a lyssavirus. Virus-neutralization assays indicated DUVV-neutralizing antibodies in 30% of the sera collected from this population of N. thebaica, providing the first indication of a Duvenhage virus circulating in this particular species and the first evidence of a bat lyssavirus circulating in Swaziland bats.