Patients with infection from bacteremic Bartonella spp., tested using Bartonella Alphaproteobacteria growth medium (BAPGM), were retrospectively categorized into one of two groups that included those whose blood was collected once (group 1; n = 55) or three times (group 2; n = 36) within a 1-week period. Overall, 19 patients (20.8%) were PCR positive for one or more Bartonella spp. using the BAPGM platform. Seven patients (12.7%) in group 1 tested positive, and 12 patients (33.3%) in group 2 tested positive. Detection was improved when the patients were tested three times within a 1-week period (odds ratio, 3.4 [95% confidence interval, 1.2 to 9.8]; P = 0.02). Obtaining three sequential blood samples during a 1-week period should be considered a diagnostic approach when bartonellosis is suspected.