Tuberculosis (TB) prevention in prisons remains a problem that requires advocacy and partnership action. A correctional officer (CO) is responsible for enforcing the rules and maintaining routines at a prison and has the authority to support TB prevention under the limitations of health manpower in prisons. The objective of this cross-sectional study was to determine the health literacy (HL) and practices of TB prevention and their association among Thailand’s COs. A total of 208 COs participated using a random sampling method. A self-administered questionnaire on HL and TB prevention practices was used for data collection. Descriptive statistics, Pearson’s chi-square test, and binary logistic regression were used for the association analysis. The majority of the participants were male (71.2%), married (60.1%), had a bachelor’s degree (60.6%), and had never been trained in TB prevention (90.9%). In total, 63.0% had adequate HL, whereas 78.4% had good practices, and this corresponded with personal prevention (75.5%) and work prevention (74.6%). Significant associations were identified for education, and communication, decision-making, and self-management skills (p < 0.05). The probability (adjusted odds ratio [95% CI]) of good practices was higher among participants with adequate communication skills (7.92 [2.15–29.24]), adequate decision-making skills (6.00 [1.86–19.36]), bachelors’ degree or higher-level education (3.25 [1.12–9.39]), and adequate self-management skills (2.95 [1.08–8.11]). The study findings show that most of the COs have adequate HL which is associated with good practices in TB prevention. Prisons should support HL development among COs for partnership and sustainable TB prevention under the constraint of health personnel.