Human infestation with head lice, Pediculus humanus capitis De Geer, is the most prevalent ectoparasitic condition in the modern world. The purpose of this study was to test human head lice from Madagascar for infection with 2 louse-borne bacteria, Bartonella quintana and Acinetobacter spp. including Acinetobacter baumannii, to assess the potential risk of exposure to these pathogens in rural populations experiencing head-louse pediculosis. A second aim was to determine the occurrence of a biomarker for permethrin resistance in head lice from 6 isolated human communities in Madagascar. Deoxyribonucleic acid (DNA) of B. quintana was detected using species-specific Fab3 gene TaqMan in 12.6% of lice from 4 villages. DNA of Acinetobacter spp. was detected using rpoB TaqMan in 42.1% of lice collected from all locations; 58.3% of rpoB-positive lice had the blaOXA51-like enzyme gene specific for A. baumannii. The kdr-resistant allele was detected in 70% of lice tested and was found in lice from each location. These results provide the first information regarding these combined characteristics of head-louse infestations in Madagascar. This approach can be applied to larger and broader surveys of lice from pediculosis capitis occurring in other geographic locations.