Background: Patients with Tuberculosis (TB) are a vulnerable group for acquiring HIV infection. Therefore, countries with a concentrated HIV epidemic and high prevalence of TB should provide adequate information about HIV prevention to TB patients. Methods: We conducted a cross-sectional study to evaluate the level of knowledge on HIV prevention and transmission among newly diagnosed TB patients in Lima, Peru. The survey evaluated knowledge about HIV infection and prevention and was administered before HIV counseling and blood sampling for HIV testing were performed. Results: A total of 171 TB patients were enrolled; mean age was 31.1 years, 101 (59%) were male. The overall mean level of knowledge of HIV was 59%; but the specific mean level of knowledge on HIV transmission and prevention was only 33.3% and 41.5%, respectively. Age and level of education correlated with overall level of knowledge in the multivariate model (P-value: 0.02 and <0.001 respectively). Conclusions: The study shows inadequate levels of knowledge about HIV transmission and prevention among newly-diagnosed TB patients in this setting, and underscores the need for implementing educational interventions in this population.