Introduction: Much of the international community has an increased awareness of potential biologic, chemical, and nuclear threats and the need for physicians to rapidly acquire new knowledge and skills in order to protect the public's health. The present study evaluated the educational effectiveness of an online bioterrorism continuing medical education (CME) activity designed to address clinical issues involving suspected bioterrorism and reporting procedures in the United States. Methods: This was a retrospective survey of physicians who had completed an online CME activity on bioterrorism compared with a nonparticipant group who had completed at least 1 unrelated online CME course from the same medical school Web site and were matched on similar characteristics. An online survey instrument was developed to assess clinical and systems knowledge and confidence in recognition of illnesses associated with a potential bioterrorism attack. A power calculation indicated that a sample size of 100 (50 in each group) would achieve 90% power to detect a 10% to 15% difference in test scores between the two groups. Results: Compared with nonparticipant physicians, participants correctly diagnosed anthrax (p = 0.01) and viral exanthem (p = 0.01), but not smallpox, more frequently than nonparticipants. Participants knew more frequently than nonparticipants who to contact regarding a potential bioterrorism event (p = 0.03) Participants were more confident than nonparticipants about finding information to guide diagnoses of patients with biologic exposure (p = 0.01), chemical exposure (p = 0.02), and radiation exposure (p = 0.04). Discussion: An online bioterrorism course shows promise as an educational intervention in preparing physicians to better diagnose emerging rare infections, including those that may be associated with a bioterrorist event, in increasing confidence in diagnosing these infections, and in reporting of such infections for practicing physicians.