Interest in Bordetella holmesii is increasing, but very little is known about this bacterium, which can be isolated from both blood and respiratory samples. In this study, we compared a B. holmesii isolate from the blood sample of an adult with bacteremia with another isolate from a nasopharyngeal swab from an adult with whooping cough syndrome. Genetic analysis was carried out, targeting relevant genes, and virulence properties were studied in cellular and animal models. Our genomic analysis provided no evidence of traits specific to either blood or respiratory isolates of B. holmesii. Neither isolate was cytotoxic to human tracheal epithelial cells. Both isolates were only weakly invasive and they did not persist within epithelial cells for less than 48 h.