Tuberculosis (TB) represents a health problem in Colombia, and its control is focused on the search for contacts and treatment of TB cases underscoring the role of latent tuberculosis infection (LTBI) as a reservoir of Mycobacterium tuberculosis . The burden of LTBI in Colombia is unknown. We aimed to estimate the prevalence of LTBI and identify the associated risk factors. In this cross-sectional study, we recruited participants from four health care centers in Cali, Colombia. The participants were eligible if they were aged between 14 and 70 years, and all participants answered a survey evaluating their medical history and sociodemographic and lifestyle factors. LTBI status was based on tuberculin skin test (TST) positivity using two thresholds: ≥10 mm (TST-10) and ≥15 mm (TST-15). The magnitude of the associations between independent factors and dependent outcomes (LTBI status and TST induration) were evaluated by logistic regression and generalized linear models, respectively. A total of 589 individuals were included with TST positivity rates of 25.3% (TST-10) and 13.2% (TST-15). Logistic regression showed that being between age 40 and 69 years (OR = 7.28, 95% CI [1.62–32.7]), being male (OR = 1.71, 95% CI [1.04–2.84]), being employed (OR = 1.56, 95% CI [1.02–2.38]), and having a low intake of alcohol (OR = 2.40, 95% CI [1.13–5.11]) were risk factors for TST positivity, while living in the north zone (OR = 0.32, 95% CI [0.18–0.55]), living in the suburb zone (OR = 0.28, 95% CI [0.15–0.52]) and having a secondary education (OR = 0.49 95% CI [0.29–0.83]) lowered the risk of TST positivity. The generalized linear model showed that the previous predictors, as well as a low body mass index, had an effect on TST reaction size. The LTBI prevalence found in the population was moderate, reflecting the continuous transmission of M. tuberculosis . Social factors seem to play a decisive role in the risk of LTBI. Employed males, who are over 40 years of age, are overweight, have a lower level of education and have a low intake of alcohol (50–100 mL, once/week) should be a priority group for prophylactic treatment as a strategy for TB control in this city.