Background: Human body lice (Pediculus humanus humanus) is recognized as the vector of different pathogenic bacteria as Bartonella quintana and Rickettsia prowazekii, the etiological agents of trench fever and epidemic typhus, respectively, being these bacteria re-emerging pathogens in different countries. Poor living conditions and limited access to public services are predisposing factors to increase high prevalence of body lice infestation in different populations such as homeless people. In Bogota-Colombia, approximately 9.614 homeless people are living in unsanitary conditions. However, our country has no reports about the presence of louse-borne bacteria and infection caused by this. Therefore, the aim of this study was to establish the presence of Bartonella spp and Rickettsia spp in body lice collected from homeless people in Bogota. Methods & Materials: A total of 201 body lice were collected from 18 individuals. Lice were taxonomically classified and pooled by host and development stage. 39 pools were obtained for the DNA extraction and all samples were subjected to PCR. For the identification of Bartonella spp, PCR was made to amplify a part of the citrate synthase (gltA) and ITS1 genes and for Rickettsia spp we amplified the gltA, ompB and 16SrRNA genes. Results: A total of 11 (28.2%) Bartonella spp lice pools were positives, from these 7 were positive for gltA, 10 positive for ITS1 and 6 positive for both genes. Also, 34 lice pools (87.1%) were positives for Rickettsia spp (gltA, ompB and 16SrRNA genes were identified 42 [62%], 1 [3%] and 17 [44%] pools positives, respectively), 6 pools positives for two genes (gltA and 16SrRNA) and 1 positive for three genes. Furthermore, 9 lice pools (23%) were positives for both microorganisms. 9 (50%) and 17 (94%) homeless individuals were positives for Bartonella spp and Rickettsia spp, respectively. While 7 (38.8%) were positives for both microorganism. Conclusion: This is the first report in Colombia and South America detecting Bartonella spp and Rickettsia spp in human body lice from homeless people, indicating that they are present in this population and create a risk for public health in Colombia, especially in susceptible populations with poor living conditions.