Background Antimicrobial stewardship programmes aim to optimise use of antibiotics and are now mandatory in all Australian hospitals. Aim We aimed to identify barriers to and enablers of appropriate antimicrobial prescribing among hospital doctors. Methods Two paper-based and one web-based surveys were administered at three Australian university teaching hospitals from March 2010 to May 2011. The 18-item questionnaire recorded doctors' level of experience, their knowledge regarding the use of common antimicrobials and their attitudes regarding antimicrobial prescribing. Local survey modifications allowed inclusion of specific questions on: infections in intensive care unit patients, clinical microbiology and use of local guidelines. Results The respondents (n = 272) were comprised of 96 (35%) registrars, 67 (25%) residents, 57 (21%) interns and 47 (17%) consultant hospital doctors. Forty-one percent were working in a medical specialty. Identified barriers included: gaps in antimicrobial prescribing knowledge (especially among interns), a lack of awareness about which antimicrobials were restricted and a reliance on senior colleagues to make antimicrobial prescribing decisions. Enablers of optimal prescribing included: an acknowledgement of the need for assistance in prescribing and reported readiness to consult national prescribing guidelines. These results were used to help guide and prioritise interventions to improve prescribing practices. Conclusion A transferable knowledge and attitudes survey tool can be used to highlight barriers and facilitators to optimal hospital antimicrobial prescribing in order to inform tailored antimicrobial stewardship interventions.