Louse flies or deer keds, Lipoptena spp., are widespread in Neotropical cervids, but the vector-borne pathogens of louse flies had only been previously reported in the Northern hemisphere. This is the first report of Bartonella spp. in deer louse flies (Lipoptena mazamae) in the neotropics collected from gray brocket deer (Mazama gouazoubira) in Southern Brazil. DNA from Bartonella sp. was detected in all 429 L. mazamae collected from 11 road-killed gray brocket deer. The same sequences of DNA of Bartonella spp. were identified in samples. Gray brocket deer are abundant in Brazil, so Bartonella-infected Lipoptena spp. may be widely distributed in the neotropics.