Bats are a significant reservoir for numerous pathogens, including Bartonella spp. It is one of the emerging zoonotic bacterial diseases that can be transmitted to humans and may cause various unspecific clinical manifestations. Thus, bartonellosis is rarely diagnosed and is regarded as a neglected vector-borne disease (VBD). Bat flies have been hypothesised to be a vector in the transmission of pathogens among bats. They are host-specific, which reduces the likelihood of pathogen transmission across bat species; however, they are likely to maintain high pathogen loads within their host species. To explore the presence of Bartonella spp. in bat flies from Peninsular Malaysia; bat fly samples collected from various sites at the east coast states were subjected to molecular detection for Bartonella spp. It was discovered that 38.7 % of bats from Terengganu and Kelantan were infested with bat flies; however, no bat fly was found in bats collected from Pahang. The collected bat flies belonged to the families Nycteribiidae (79.6 %) and Streblidae (20.4 %). The collected bat flies were pooled according to the locations and species into 39 pools. Out of these 39 pools, 66.7 % (n = 26) were positive for Bartonella spp. by PCR. Sequence analyses of five randomly selected PCR-positive pools revealed that pools from Kelantan (n = 3) have the closest sequence identities (99 %) to Bartonella spp. strain Lisso-Nig-922 from Nigeria. However, the other pools from Terengganu (n = 2) were closely related to Bartonella spp. strain KP277 from Thailand and Bartonella spp. strain Rhin-3 from the Republic of Georgia with 99 % and 100 % sequence identity, respectively. This suggests that the Bartonella spp. found in Malaysian bat flies are genetically diverse and can potentially serve as reservoirs for pathogenic Bartonella spp.