Ticks, belonging to the soft ticks species Ornithodorus sonrai, have been collected from six sites in Senegal and were tested for the presence of Bartonella spp. Initial screening by PCR revealed the presence of these bacteria in ticks from two villages, Soulkhou Thissé (5/8, 62.5%) and Maka Gouye (1/24, 4.2%). Three bacterial strains were isolated from live ticks, and the genetic characterization of these strains suggests that they belong to two previously unknown species. The pathogenicity of these two new species of Bartonella is not yet known. The new isolates described here are the first strains of Bartonella spp. from soft ticks and the first isolates from any arthropod species in Africa.