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We captured, ectoparasite-combed, and blood-sampled cave-roosting Madagascan fruit bats (Eidolon dupreanum) and tree-roosting Madagascan flying foxes (Pteropus rufus) in four single-species roosts within a sympatric geographic foraging range for these species in central Madagascar. We describe infection with novel Bartonella spp. in sampled Eidolon dupreanum and associated bat flies (Cyclopodia dubia), which nest close to or within major known Bartonella lineages; simultaneously, we report the absence of Bartonella spp. in Thaumapsylla sp. fleas collected from these same bats. This represents the first documented finding of Bartonella infection in these species of bat and bat fly, as well as a new geographic record for Thaumapsylla sp. We further relate the absence of both Bartonella spp. and ectoparasites in sympatrically sampled Pteropus rufus, thus suggestive of a potential role for bat flies in Bartonella spp. transmission. These findings shed light on transmission ecology of bat-borne Bartonella spp., recently demonstrated as a potentially zoonotic pathogen. Author Summary: Bartonella spp. are bacteria that inhabit the red blood cells of both human and animal hosts. Among humans, Bartonella spp. are known to cause several febrile illnesses, including Carrion's disease (Bartonella bacilliformis), trench fever (Bartonella quintana), and cat scratch fever (Bartonella henselae), all of which are transmitted via arthropod vectors—respectively sand flies, lice, and fleas. Bats are known to host multiple Bartonella spp., including some capable of infecting humans. Some bat species are also known to host obligate ectoparasites known as bat flies (Diptera: Hippoboscoidea), which also sometimes support Bartonella spp. infections. The role of bat flies and other bat ectoparasites as vectors for Bartonella spp. transmission has been suggested, but not fully explored. We demonstrate Bartonella spp. infection in one species of Madagascar fruit bat, which hosts bat flies, simultaneously with the absence of Bartonella in a fruit bat species of overlapping range that appears not to support these ectoparasites. In light of ongoing trends of zoonotic emergence of human diseases from bat reservoirs, further understanding of the transmission dynamics of bat-borne pathogens is paramount. [ABSTRACT FROM AUTHOR]