Objective The aim of this study was to identify perceived barriers to appropriate antibiotic prescribing across different healthcare settings. Methods A cross-sectional survey of clinicians in Australian hospitals and primary care was undertaken between June and October 2019. The perceived barriers to appropriate antibiotic prescribing were considered as dependent variables, whereas age, sex, clinical experience, healthcare setting and the use of guidelines were considered independent variables. We used multivariate logistic regression to identify factors predictive of inappropriate antibiotic prescribing. Content analysis of free-text responses provided additional insights into the impediments to appropriate prescribing. Results In all, 180 clinicians completed the survey. Overall, diagnostic uncertainty and limited access to guidelines and prescribing information were significant barriers to appropriate antibiotic prescribing. Factors associated with these barriers were clinical experience, care setting (hospitals vs primary care) and the use of guidelines. Experienced clinicians (>11 years) were less likely to consider that limited access to information negatively affected prescribing practices (experience 11-20 years, odds ratio (OR) 0.66, 95% confidence interval (CI) 0.31-0.84; experience >20 years, OR 0.51, 95% CI 0.24-0.91). Conversely, general practitioners considered diagnostic uncertainty (OR 1.31, 95% CI 1.09-1.63) and patient expectations (OR 1.41, 95% CI 1.12-1.84) were more likely to be perceived barriers to appropriate prescribing. The use of guidelines and clinical experience may counteract this. Conclusion Years of experience, use of guidelines and type of setting were predictors of clinicians' perceptions regarding antibiotic prescribing. Our data highlight the importance of individual and setting characteristics in understanding variations in prescribing practices and designing targeted interventions for appropriate antibiotic prescribing. What is known about the topic? Inappropriate antibiotic prescribing is a significant health issue in Australia. Drivers of inappropriate prescribing are known, but how individual and setting characteristics contribute to variations in prescribing behaviour has not been fully understood. What does this paper add? Diagnostic uncertainty and limited access to prescribing information, including guidelines, formulary restrictions and antibiotic resistance patterns, can limit appropriate antibiotic prescribing. Clinicians' years of experience, the healthcare settings and clinician use of guidelines are important predictors of antibiotic prescribing behaviour. What are the implications for practitioners? The findings of this study can inform the design of tailored interventions to promote rational antibiotic prescribing practices in general practice and hospital settings.