BACKGROUND: Because of the association of mental health and substance use disorders with higher HIV mortality and decreased retention in care, we investigated their frequency and impact on tuberculosis preventive therapy (TPT) adherence and completion among people with HIV (PWHIV) initiating TPT. METHODS: We conducted a prospective, longitudinal cohort study with a nested mixed methods study in 2 Johannesburg, South Africa, facilities. Participants were PWHIV on antiretroviral therapy initiating TPT between August and December 2023. We measured TPT adherence and completion with electronic medication boxes. We used validated tools to measure symptoms of anxiety, depression, alcohol use, and other substance use at enrollment and 12 weeks. We constructed multivariable regression models to determine associations of these variables with TPT adherence and completion, adjusting for age, sex, and time on antiretroviral therapy. We interviewed participants about mental health and experiences with TPT and analyzed responses using deductive content analysis. RESULTS: Among 224 PWHIV, 111 (50%) completed TPT. Eighty-one (36%) screened positive for depression symptoms and 63 (28%) for anxiety symptoms. Seventy-six (34%) reported unhealthy alcohol use and 60 (27%) tobacco use. Using multivariable models adjusting for confounders, only depression symptoms were significantly and negatively associated with adherence (9% fewer doses; 95% confidence interval, .4-16; = .032) and completion (odds ratio, 0.48; 95% confidence interval, .26-.90; = .021). Participant narratives highlighted the negative influence of mental health on adherence and the need for social and psychological support services. CONCLUSIONS: Symptoms of depression, anxiety, unhealthy alcohol use, and tobacco use were common among PWHIV initiating TPT. Depression symptoms were strongly and independently associated with TPT nonadherence and noncompletion.