This study assessed community health workers' (CHW) knowledge on tuberculosis (TB), TB control measures, and directly observed therapy (DOT) in Belo Horizonte, Minas Gerais State, Brazil. This was a cross-sectional study that applied a questionnaire in a random and representative sample of 489 CHW. The overall proportion of correct answers was 74.6%. The mean proportions of correct answers in the domains for TB, TB control measures, and DOT were 81%, 84.1%, and 59.4%, respectively. The study showed knowledge gaps in the identification of patients with pulmonary TB, the target public for DOT, and adequate technique for treatment supervision. Better knowledge was associated with time working as a CHW (OR = 2.3) and follow-up of cases in the 12 months prior to the interview (OR = 1.7). The study provided new scientific data on level of knowledge among CHW concerning DOT and can support strategies to improve TB control activities.