In the present study, we tested 391 fleas collected from Guinea pigs (Cavia porcellus) (241 Pulex species, 110 Ctenocephalides felis, and 40 Tiamastus cavicola) and 194 fleas collected from human bedding and clothing (142 Pulex species, 43 C. felis, five T. cavicola, and four Ctenocephalides canis) for the presence of Bartonella DNA. We also tested 83 blood spots collected on Flinders Technology Associates (FTA) cards from Guinea pigs inhabiting 338 Peruvian households. Bartonella DNA was detected in 81 (20.7%) of 391 Guinea pig fleas, in five (2.6%) of 194 human fleas, and in 16 (19.3%) of 83 Guinea pig blood spots. Among identified Bartonella species, B. rochalimae was the most prevalent in fleas (89.5%) and the only species found in the blood spots from Guinea pigs. Other Bartonella species detected in fleas included B. henselae (3.5%), B. clarridgeiae (2.3%), and an undescribed Bartonella species (4.7%). Our results demonstrated a high prevalence of zoonotic B. rochalimae in households in rural areas where the research was conducted and suggested a potential role of Guinea pigs as a reservoir of this bacterium.