Background Pre-exposure prophylaxis (PrEP) involves administering antiretroviral drugs to prevent human immunodeficiency virus (HIV) infection in at-risk subjects. Chile is considered one of the countries with the highest number of new cases per year of HIV infections. Methods A nationwide cross-sectional study was carried out in Chile. A questionnaire of physicians' attitudes toward the prescription of PrEP was used. Results 632 doctors responded correctly the survey. 58.5% ( n = 370) were women, and median age was 34 years (IQR 25–43). 55.4% ( n = 350) responded that they have never prescribed antiretrovirals for HIV-negative individuals to prevent HIV infection, and only 10.1% have prescribed PrEP. 60.8% ( n = 384) mentioned having informed about the possibility of using antiretroviral post-exposure prophylaxis in case of risky sexual activity. 76.3% ( n = 482) believed each institution should formulate internal protocols for administering these drugs, and 98.4% ( n = 622) stated that with the currently available evidence, PrEP should be suggested to cope with the HIV pandemic. Conclusion It was concluded that knowledge, attitudes and experience toward PrEP prescribing are variable and related to patient care. However, Chile has a marked tendency in favor of this therapy, which is similar to that reported in studies worldwide.