Bats, with their robust immune system, frequently serve as hosts for viruses and bacteria. Bartonella spp. are transmitted by blood-sucking arthropods and are widely distributed among various mammalian species globally. The present study focused on the molecular detection of Bartonella spp. From ectoparasites infesting widely distributed Rhinolophus affinis in Southwest China. Briefly, the bats were captured from four districts, namely Xundian, Jinning, Lufeng, and Mouding and ectoparasites collected from these bats. The ectoparasites were identified based on morphological characteristics, and the biodiversity of ectoparasites infesting bats in the study area was elucidated through spatial distribution and analysis of dominant species. Further, DNA was extracted from ectoparasites and three targeted genes (ftsZ, gltA, and rpoB) of Bartonella were amplified using conventional PCR. Sixty R. affinis bats were captured with an impressive ectoparasites infestation rate of 90.00%. A total of 10 species were identified comprising 1,412 ectoparasites, with the dominant species being Eyndhovenia euryalis (Y = 0.099, m*/m = 2.439), Macronyssys tieni (Y = 0.563, m*/m = 2.040), Macronyssys dechangensis (Y = 0.024, m*/m = 1.815), Ixodes vespertilionis (Y = 0.026, m*/m = 2.146), and Stylidia fraterna (Y = 0.052, m*/m = 1.436) (one species of bat fly), all of which exhibited aggregated distribution on the host's body surface. Notably, the detection of 172 ectoparasites in the bats revealed that all positive cases of Bartonella were found in S. fraterna. This high prevalence may be attributed to the unique reproductive strategy of bat flies, which facilitates vertical transmission of Bartonella. Therefore, bat flies may act as potential vectors for Bartonella transmission. This study deepens understanding of the ectoparasite diversity of R. affinis and broadens our knowledge of the geographical distribution of this pathogen.