Introduction: Post-exposure prophylaxis (PEP) is the use of antiretroviral drugs (ARVs) to reduce the risk of human immunodeficiency virus (HIV) infection after potential risk exposure. ARV-based interventions are recommended as part of combination HIV prevention, especially for key populations. Objective: The aim of this study was to measure knowledge about PEP among university students. Methods: A cross-sectional study was conducted on university students from the Health, Education, Exact, and Human Sciences Departments of the State University of Bahia, Brazil. Sociodemographic data, information on sexual behavior, and knowledge of PEP were collected through a standardized self-applied questionnaire. Results: We analyzed 1580 questionnaires, of which 66.7% (1024/1536) were from females, with a mean age of 23.9 (±6.5) years, and 35.4% (448/1264) reported irregular use of condoms and regular use was not associated with being students from the health area (p=0.44, OR 0.90, 95%CI 0.69–1.17). Regarding PEP, 28.5% (449/1578) had known about it and their knowledge was statistically associated with men who have sex with men (MSM) (p<0.01, OR 3.92, 95%CI 2.45–6.28). It was noted that 94.0% (1485/1579) did not know the time limit for starting PEP, 95.1% (1500/1578) did not know the duration of prophylaxis, and 91.1% (1437/1577) did not know where to get PEP. Finally, 0.4% (7/1578) referred to previous use and 96.6% (1488/1540) would not change their sexual behavior after knowing about PEP. Conclusion: PEP is a prevention strategy available for decades and is safe, effective, and cost-effective. However, it is underutilized and a lack of knowledge on PEP is one of the main obstacles to access. Among university students, there is a limited knowledge about PEP acting as a barrier in preventing new infections, which shows the need for interventions based on sexual-health education, stimulating the reduction of risk behaviors and disseminating information about combination prevention.