Purpose: To determine the prevalence and demographic risk factors for latent tuberculosis infection (LTBI) among healthcare workers in Semarang, Indonesia. Methods: A cross-sectional study involving 195 healthcare workers from 34 primary health centers was conducted from August to October 2019. The relationship between independent variables and dependent variables was analyzed using a multivariable logistic regression analysis. Results: The prevalence of LTBI among healthcare workers in this study was 23.6%. Comorbidities were the only risk factor for LTBI identified among other risk factors (OR=3.39, 95% CI: 0.99-11.62, p=0.04). Other demographic factors such as age (OR=0.93, 95% CI: 0.45-1.92, p=0.839), gender (OR=0.79, 95% CI: 0.23-2.72, p=0.708, smoking habits (OR=2.54, 95% CI: 0.52-12.38, p=0.247), and length of work (OR=1.43, 95% CI: 0.70-2.91, p=0.331) were not significant risk factors for LTBI. Conclusion: Healthcare workers suffering from comorbidity have a high risk for tuberculosis infection, and should not work in areas where they would be exposed to patients with tuberculosis. Healthcare workers need to apply occupational safety standards during contact with TB patients or specimens to minimize the disease transmission.