TheBartonellagenus consists of neglected pathogens associated with potentially transfusional-transmitted and fatal human diseases. We aimed to evaluateBartonellasp. prevalence in 500 blood donors and compare the results with the data already published about these samples. We used molecular diagnostic methods to detectBartonellasp.-DNA from blood and liquid culture samples: (A) conventional PCR for two gene regions, the ITS targeting the genusBartonellaand the specificgltA Bartonella henselae; (B) nested PCR for theftsZgene and (C) qualitative real-time PCR for thegltAgene, bothB.henselaespecific. We obtained 30/500 (6%) DNA detections from the blood samples; 77/500 (15.4%) DNA detections from liquid culture samples and five (1%) samples had DNA detection from both. In total, we detectedB.henselaeDNA from 102/500 (20.4%) donors. The samples used in this study had already been submitted forBartonellasp.-DNA detection using only a conventional PCR in liquid culture. Sixteen samples (3.2%) were positive previously, and from these 16 samples, 13 were negative in the new investigation. We concluded that the use of liquid culture combined with different molecular tests increases the possibility of detectingBartonellasp.-DNA, but the tests do not avoid false-negative results. More than a fifth of blood donors had at least one PCR that detectedBartonellasp.-DNA among the eight molecular reactions performed now (four reactions in whole blood and four in liquid culture). Seven percent hadB.henselae-DNA detection for two or more distinct regions. Considering the results obtained previously, the DNA ofBartonellaspp. was detected or the agent isolated in 23% of analyzed blood donors. The results establish that the low bacteremia and the fastidious characteristics of the bacterium are challenges to laboratory diagnosis and can make it difficult to confirm the infection in patients with bartonelloses.