Health care workers are at increased risk of nosocomial transmission of tuberculosis (TB). This study aimed to determine the prevalence of TB among health care workers and its associated factors. A retrospective analysis was conducted using database of TB screening program at Hospital Canselor Tuanku Mukhriz (HCTM) over the period of three years (2018-2020). The prevalence rate of TB showed increasing trend, from 1.54 per 100 health care workers in 2018 to 5.02 per 100 health care workers in 2020. Aged group of 31-40 had higher risk for contracting TB (p<0.001), health support workers had significant association with TB acquisition (p=0.008), workers in Diagnostic Laboratory Department had the high risk of getting infected with TB (p<0.001) and the workplace with the highest prevalence was in laboratory (45.9%). In terms of TB index cases among health care workers, 16 cases (69.5%) were pulmonary tuberculosis (PTB), 6 cases (26.1%) were extrapulmonary tuberculosis (EPTB) and 1 case (4.4%) had both PTB and EPTB. It was crucial for health care workers to be aware of these significant associations and for relevant authorities to carry out protective measures.