BACKGROUND: Room ventilation is a key determinant of airborne disease transmission. Despite this, ventilation guidelines in hospitals are not founded on robust scientific evidence related to the prevention of airborne transmission. METHODS: We sought to assess the effect of ventilation rates on influenza, tuberculosis, and rhinovirus infection risk within 3 distinct rooms in a major urban hospital: a lung function laboratory, an emergency department negative-pressure isolation room, and an outpatient consultation room. Air-exchange rate measurements were performed in each room using CO2 as a tracer. The model developed by Gammaitoni and Nucci was used to estimate infection risk. RESULTS: Current outdoor air-exchange rates in the lung function laboratory and emergency department isolation room limited infection risks to 0.1%-3.6%. Influenza risk for individuals entering an outpatient consultation room after an infectious individual departed ranged from 3.6% to 20.7%, depending on the duration for which each person occupied the room. CONCLUSION: Given the absence of definitive ventilation guidelines for hospitals, air-exchange measurements combined with modeling afford a useful means of assessing, on a case-by-case basis, the suitability of room ventilation for preventing airborne disease transmission.