Nurses are primary responsible for Direct Observation Therapy Strategy and administration of anti-tuberculosis (TB) medications. Lack of knowledge might result with medication errors and ineffective TB control. The purpose of this study was to assess knowledge of nurses about anti-TB treatment. A descriptive cross-sectional study was conducted in Turkey with 208 nurses employed at TB and lung disease clinics of health-care settings in Ankara. Data were collected through a questionnaire, which included questions about demographics and a knowledge test with true–false questions related to TB treatment. Overall scores were high with a mean score of 18.5 out of 24, but there was knowledge deficiency in effects and side-effects of anti-TB drugs. Knowledge is foundational for any practice and for TB control. Clinical experience and continuing education after graduation influence the level of knowledge.