Tuberculosis (TB) among healthcare workers (HCWs) highly affects morbidity and TB transmission in hospitals. A retrospective cohort study of TB among HCWs (HCW-TB) was conducted using a registered database from 2003 to 2016 at Chiang Mai University Hospital to determine clinical and microbiological characteristics and treatment outcomes of HCW-TB. A total of 76 patients comprising 54 nurses (71.1%), 12 physicians (15.8%), and 10 paramedics (13.2%) were diagnosed with TB disease. The men to women ratio was 25:51, with a mean age of 37.0±11.6 years, a median work duration of 12.0 years (5–20) and a body mass index of 19.4±2.5 kg/m². Within the HCW-TB group, 28 (36.8%) worked in the Medical Department, 12 (15.8%) worked in the Outpatient Department/Emergency Room, and 9 (11.8%) worked in the Surgical Department. Pulmonary TB (PTB) was the most common manifestation of HCW-TB (92.1%). Sputum acid-fast stains were positive among 28 (40.0%) HCWs with PTB. Mycobacterium tuberculosis cultures were positive in 26 (34.2%) patients. Drug susceptibility testing showed sensitivity to all first-line drugs (75.0%), resistance to any one first-line drug (20.8%), and multidrug-resistant TB comprised 4.2%. The end-of-treatment success rate was 100%. Therefore, TB control guidelines should be strictly implemented to prevent TB transmission in healthcare settings.