Objectives: To analyze the sequence of the region involved in the development of quinolone resistance of the gyrA and parC genes in a series of Bartonella bacilliformis isolates recovered prior to the introduction of quinolones, as well as one clinical isolate recovered in the 1970s, establishing the susceptibility levels to nalidixic acid and ciprofloxacin. Methods: Five B. bacilliformis were studied: four isolated before 1957, prior to the introduction of quinolones in clinical practice. The remaining strain was isolated in 1977. A fragment of the gyrA and parC genes was amplified and sequenced. Susceptibility to nalidixic acid and ciprofloxacin was established by the E-test method. Results: All the strains were resistant to nalidixic acid (minimum inhibitory concentration (MIC) >256 mg/l). Three isolates presented decreased susceptibility to ciprofloxacin and two were highly resistant (MIC >32 mg/l). All the strains presented an Ala at position 91 of GyrA and position 85 of ParC. Conclusions: B. bacilliformis presents a constitutive resistance to quinolones, which may be related to the presence of Ala at position 91 of GyrA and 85 of ParC. These results advise against the current clinical guidelines recommending the use of ciprofloxacin to treat bartonellosis in some countries of the Andean area. © 2009 International Society for Infectious Diseases.