Background Physicians have a crucial role in the implementation and scale up of pre-exposure prophylaxis (PrEP). The objective of this study is to examine Belgian physicians’ PrEP knowledge, concerns, acceptance and their willingness to prescribe PrEP. Methods: A cross-sectional online survey was conducted between March and June 2016. Dissemination targeted Belgian primary care physicians (PCPs) and HIV specialists. Sociodemographic characteristics, experience with HIV and PrEP, self-assessed PrEP knowledge, concerns about PrEP, and PrEP acceptance were stratified according to professional background. Associations with willingness to prescribe PrEP were examined using univariable and multivariable binary logistic regression analyses. Results: In total, 333 completed surveys were included in the analysis. Sixty-two physicians (18.6%) scored their knowledge of PrEP to be good, 263 (79.0%) had an accepting attitude towards PrEP and 198 (59.5%) were willing to prescribe PrEP if approved in Belgium. HIV specialists consistently reported having better knowledge of PrEP, less concerns and a more accepting attitude towards PrEP than PCPs. In multivariable logistic regression analysis, higher PrEP knowledge (OR 2.4; 95%CI: 1.0–5.7) and higher PrEP acceptance (OR: 3.8; 95%CI: 2.1–6.8) remained significantly associated with the willingness to prescribe PrEP. Conclusions: HIV specialists are better prepared to provide PrEP than PCPs in Belgium. Interventions to improve PrEP knowledge and acceptance among all providers are needed. The role of PCPs could be very important in optimising the rollout of PrEP, but additional training and guidelines will be needed.