Context: Tuberculosis (TB) treatment support is one of the recommended strategies to enhance treatment adherence and outcomes. Treatment supporters are at risk of contracting TB and adequate knowledge of TB and good preventive practices are required for their protection. Aims: This study aimed at assessing the knowledge and preventive practices of TB treatment supporters at Directly Observed Treatment Short-course (DOTS) centers in Lagos Mainland Local Government Area of Lagos state, Nigeria. Settings and Design: This cross-sectional study was conducted among 196 TB treatment supporters selected from five DOTS centers in Lagos. Methods: Data were obtained using an adapted pretested questionnaire. Statistical Analysis Used: Bivariate and multivariate analyses were performed to determine the factors associated with self-protection practices. A P < 0.05 was considered statistically significant. Results: The mean age of the participants was 37.3 ± 12.1 years. More than half of the respondents were females (59.2%) and immediate family members (61.3%). Overall, 22.5% had good knowledge of TB, while 53.0% had positive attitudes toward TB. Only 26.0% adequately protected themselves from infection. The caregiver's level of education ( P = 0.001) and their relationship to the patient ( P = 0.001) were significantly associated with good preventive practices in bivariate analysis. Not being related to the patient was a predictor of adequate TB prevention practices (adjusted odds ratio = 2.852; P = 0.006; 95% confidence interval = 1.360–5.984). Conclusions: This study revealed low levels of TB knowledge and fair preventive practices, especially among caregivers who are relatives. There is, therefore, a need to improve population literacy about TB and its prevention and a more focused orientation of relatives who volunteer as treatment supporters, through health education, with periodic monitoring during clinic visits, of how they prevent TB.