We conducted a prospective study about sexually transmitted infections (STIs) knowledge in different populations attending Lyon's University Hospitals in order to estimate awareness on STIs. Pre-exposure prophylaxis (PrEP)-users (PrEP group), persons living with HIV (PLWH group) and persons undergoing free STI screening (screening group) filled an anonymous questionnaire evaluating STI knowledge. A composite STI knowledge score was calculated and was correlated with patients’ characteristics. A total of 756 patients were enrolled in three groups: screening (n = 509), PrEP (n = 103) and PLWH (n = 144). STI transmission knowledge was better for HIV than for other STIs. The median STI knowledge score was significantly higher in PrEP-users than in the screening and PLWH groups. PrEP use and a previous STI diagnosis were independently associated with a higher score. PrEP-users have better STI knowledge than PLWH and persons undergoing free STI screening. Sexual health promotion interventions routinely reserved to PrEP-users in France seem to be effective in raising the awareness of this group for STIs. Continuous efforts are justified for PLWH and the younger layers of the population.