Background: Tuberculosis (TB) is a treatable and curable disease. Junior doctors and nurses working in health-care settings are often the first to identify and manage TB cases. Hence, a thorough knowledge regarding TB disease and its management under Revised National Tuberculosis Control Programme (RNTCP), and recent guidelines on Directly Observed Treatment Shortcourse (DOTS) strategy is crucial for health care professionals. Objectives: To assess the knowledge regarding tuberculosis, RNTCP and DOTS among health care professionals at a tertiary care hospital. Methodology: This cross-sectional, observational, descriptive, and institution based study was done among health care professionals in a tertiary care hospital. Results: Out of 183 participants, 47.5% were junior doctors and 52.5% were nursing staffs. 79.3% of junior doctors and 58.3% of nursing staffs had good knowledge of tuberculosis. 96.6% of junior doctors had received training on RNTCP. 65.5% of junior doctors and 55.2% of nursing staffs had good knowledge regarding treatment monitoring and follow-up of patients. Conclusion: Junior doctors and nursing staffs had a good knowledge regarding TB and recent RNTCP guidelines. RNTCP sensitization should be the part of regular activity of all medical colleges and hospitals to train the health care professionals.