The concentration and size distribution of infectious aerosols produced by patients with pulmonary tuberculosis (TB) has never been directly measured. We aimed to assess the feasibility of a method that we developed to collect and quantify culturable cough-generated aerosols of Mycobacterium tuberculosis. Subjects were recruited from a referral hospital and most had multidrug-resistant TB. They coughed into a chamber containing microbial air samplers while cough frequency was measured during two 5-minute sessions. Cough-generated aerosol cultures were positive in 4 of 16 subjects (25%) with smear-positive pulmonary TB. There was a rapid decrease in the cough-generated aerosol cultures within the first 3 weeks of effective treatment. Culture-positive cough aerosols were associated with lack of treatment during the previous week (p = 0.007), and there was a trend in the association with cough frequency (p = 0.08). The size distributions of these aerosols were variable, but most particle sizes were in the respirable range. Quantification of viable cough-generated aerosols is feasible and offers a new approach to study infectiousness and transmission of M. tuberculosis and other airborne pathogens.