Background: The clinical presentation of Bordetella pertussis can overlap with that of respiratory syncytial virus (RSV), and coinfection does occur, but management differs. Hypothesis: The prevalence of B. pertussis is < 2% among Emergency Department (ED) patients with bronchiolitis. Our secondary hypothesis was that the prevalence of Bordetella parapertussis is also < 2% among these patients. Methods: Nasal washings were obtained from children up to 18 months of age (inclusive) who presented to a county hospital ED with a clinical diagnosis of bronchiolitis. These washings were frozen to −70°C before testing for B. pertussis and B. parapertussis using species-specific real-time polymerase chain reaction (PCR) assays. The assays were optimized to target conserved regions within a complement gene and the CarB gene, respectively. A Bordetella spp. genus-specific real-time PCR assay was designed to detect the Bhur gene of B. pertussis, B. parapertussis, and B. bronchiseptica. RSV antigen detection was also performed. Results: There were 227 patients enrolled. After exclusions, 204 remained in the analysis. RSV antigen testing was positive in 109/186 (59%) of the patients in whom it was performed. All samples were tested for B. pertussis. B. parapertussis testing could not be completed on 23 samples. No cases (0/204; 95% confidence interval [CI] 0–1.8%) tested positive for B. pertussis or B. parapertussis (0/181; 95% CI 0–2%). Conclusion: The prevalence of B. pertussis in children presenting to the ED with bronchiolitis was < 2%.