Malaysia is rated as having an intermediate burden of tuberculosis, with a notification rate of 78 cases per 100,000 population and a mortality rate of 7.9 per 100,000 population in 2022. Previous studies demonstrated that there were moderate to high prevalence of latent tuberculosis infection (LTBI) among the medical and nursing students. The World Health Organisation has highlighted detection, diagnosis, and treatment of LTBI as a critical strategy for TB eradication. To stop the cycle of TB transmission and infection, screening is the initial step in treating patients who have LTBI. However, with 25% of the world's population having a past infection of the Mycobacterium tuberculosis (MTB) and remaining asymptomatic, and 1 in 10 with LTBI progressing to active TB, eliminating TB must begin with an awareness of who to screen. Following this, we conducted a study to determine the prevalence and the level of knowledge, attitude, and practice (KAP) on LTBI among medical and nursing students. A cross-sectional study was conducted among the medical and nursing students via stratified random sampling. Tuberculin Skin Test (TST) and Interferon- Gamma Release Assays (IGRA) were used for prevalence of LTBI. KAP questionnaire was adapted from previous studies and data were collected via online form. A total of 345 respondents (medical students=171, nursing students =174) participated in this study. Due to movement control order during the pandemic, the LTBI prevalence study among nursing students could not be completed. The prevalence of positive TST among medical students was 3.5% and the prevalence of LTBI from IGRA test was 0.6%. The level of knowledge on LTBI among both medical and nursing students was moderate (40.9%), a positive attitude towards LTBI was high (88.1%) and a good practice on prevention of LTBI was also high (97.1%). Factors associated with higher knowledge were age (p=0.001), year of study (p=0.001), clinical exposure (p=0.003), program of study (p=0.001) and five TB exposure variables. Factors associated with positive attitude were age (p= 0.047), program of study (p=0.044), clinical exposure (p=0.001) and two TB exposure variables. Factors associated with good practice were gender (p=0.014), program of study (p=0.010) and clinical exposure (p=0.037). The prevalence of LTBI among medical students in Negeri Sembilan was low. Even though medical and nursing students have moderate knowledge on LTBI, they have a positive attitude towards LTBI and good practice in prevention of LTBI. Active surveillance for TB and LTBI among medical and nursing students should be implemented routinely for early detection and intervention.