Background Pre-exposure prophylaxis (PrEP) is a safe and effective medication for preventing HIV acquisition. We examined Australian general practitioners' (GP) knowledge of PrEP efficacy, characteristics associated with ever prescribing PrEP and barriers to prescribing. Methods We conducted an online cross-sectional survey of GPs working in Australia between April and October 2022. We performed univariable and multivariable logistic regression analyses to identify factors associated with: (1) the belief that PrEP was at least 80% efficacious; and (2) ever prescribed PrEP. We asked participants to rate the extent to which barriers affected their prescribing of PrEP. Results A total of 407 participants with a median age of 38years (interquartile range 33-44) were included in the study. Half of the participants (50%, 205/407) identified how to correctly take PrEP, 63% (258/407) had ever prescribed PrEP and 45% (184/407) felt confident with prescribing PrEP. Ever prescribing PrEP was associated with younger age (AOR 0.97, 95% CI: 0.94-0.99), extra training in sexual health (AOR 2.57, 95% CI: 1.54-4.29) and being a S100 Prescriber (OR 2.95, 95% CI: 1.47-5.90). The main barriers to prescribing PrEP included: 'Difficulty identifying clients who require PrEP/relying on clients to ask for PrEP' (76%, 310/407), 'Lack of knowledge about PrEP' (70%, 286/407) and 'Lack of time' (69%, 281/407). Conclusion Less than half of our GP respondents were confident in prescribing PrEP, and most had difficulty identifying who would require PrEP. Specific training on PrEP, which focuses on PrEP knowledge, identifying suitable clients and making it time efficient, is recommended, with GPs being remunerated for their time.