Background Expanded access to HIV pre-exposure prophylaxis (PrEP) is being actively debated in Australia. Awareness and knowledge of this HIV-prevention method have not been assessed in detail in the primary affected population, gay and bisexual men. Methods: Awareness and knowledge of PrEP were assessed among Australian gay and bisexual men, who were asked to complete a national, anonymous, online survey in 2015. Associations with PrEP awareness were identified with multivariate logistic regression and associations with PrEP knowledge were identified using multivariate linear regression. Results: Among 1251 participants, 954 (77%) were aware of PrEP. The most common sources of information were gay community media, Australian websites and friends. Awareness of PrEP was independently associated with older age, living in a capital city, having a university degree, being tested for HIV, being HIV-positive, having condomless anal intercourse with regular male partners, and ever having taken post-exposure prophylaxis. Men in monogamous relationships were less likely to be aware of PrEP. Among men who were aware of PrEP, the mean PrEP knowledge score was 6.8 out of 13. Relatively few participants knew that taking PrEP involved regular clinical monitoring and that in Australia PrEP was only recommended for people at risk of HIV. Better knowledge was independently associated with living in a capital city, having a university degree, being in full-time employment, being HIV-positive, and ever having taken post-exposure prophylaxis or PrEP. Conclusions: To assist in appropriate PrEP uptake, we recommend educating gay and bisexual men about current Australian prescribing guidelines and how PrEP is accessed in Australia.