Introduction: Bartonella bacilliformis is the etiological agent of Carrion’s disease, which is a neglected disease linked to people in low-socioeconomic populations in Andean valleys. An outbreak of B. bacilliformis was reported in a rural area of the Peruvian Amazon region. The aim of this study was to characterize this outbreak using molecular techniques. Methodology: Fifty-three blood samples from patients diagnosed with Carrion’s disease were analyzed by molecular tools, using both a Bartonella-specific polymerase chain reaction (PCR) and an universal PCR, both based on 16S rRNA gene amplification. Additional water samples from the area were also analyzed. Results: Unexpectedly, the samples were positive only when the universal PCR was used. Although environmental contamination cannot be ruled out, the results showed that Sphingomonas faeni was the possible causative agent of this outbreak, and that water was the most feasible infection source. Conclusions: Diagnosis by clinical criteria or microscopy may lead to misdiagnosis. There is a need to include molecular tools in the routine diagnosis of febrile syndromes, including Carrion’s disease.