Bats are known reservoir hosts for a wide variety of parasites and pathogens, including bacteria and protozoans. Some of these pathogens are vector-borne, and although their role is poorly studied, ectoparasites may contribute significantly to their transmission. The aim of this study was to molecularly detect the presence of vector-borne microorganisms in bat-associated ectoparasites to explore their diversity and distribution in these insects. We tested the presence of Bartonella spp., Polychromophilus spp., and Trypanosoma spp. in bat flies and bat fleas collected from 56 Egyptian Rousette bats (Rousettus aegyptiacus), using conventional PCR. We found a high prevalence of 43.9% (47/107) of Bartonella spp. in bat flies, but a low prevalence of 6.6% (4/61) in bat fleas. Polychromophilus and Trypanosoma DNA were absent in both bat flies and bat fleas. Furthermore, we found novel gltA Bartonella sequences, as well as genotypes that are highly similar to recently described and potentially zoonotic ones. Our results show high diversity of Bartonella in bat flies, however, their role in pathogen transmission is still unknown and should be further explored.