Although many studies have assessed factors affecting culture conversion during tuberculosis treatment, few have looked into the effect of tobacco smoking. This study included 89 active pulmonary tuberculosis patients with positive sputum culture upon presentation and collected information regarding smoking history and culture conversion after 60 days of therapy. Current smokers had a higher risk (OR 5.6; 95%CI 1.7–18.7) of non-conversion after two months of therapy when compared to never and ex-smokers. Cavities on chest X-ray and alcohol abuse were shown to confound this association. After adjustment for cavities on the chest X-ray and alcohol abuse current smoking compared to current non-smoking remained significantly associated with culture non-conversion at 60 days of treatment (adjusted OR 6.9; 95%CI 1.8–26.7, p = 0.002) with a significant (p = 0.004) trend in adjusted OR with the number of cigarettes smoked daily to 11.6 (1.8–73.4) among those smoking more than 20 cigarettes per day. In conclusion tobacco smoking was found to delay culture conversion during treatment for pulmonary tuberculosis in a dose-dependent manner. More research is needed to elucidate the effects of smoking on tuberculosis treatment response, and of smoking cessation during tuberculosis treatment.