Abstract Background Understanding physicians’ antibiotic-prescribing behaviour is fundamental when it comes to improving antibiotic use and tackling the growing rates of antimicrobial resistance. The aim of the study was to develop and validate -in terms of face validity, content validity and reliability- an instrument designed to assess the attitudes and knowledge underlying physician antibiotic prescribing. Methods The questionnaire development and validation process comprised two different steps, namely: (1) content and face validation, which included a literature review and validation both by physicians and by Portuguese language and clinical psychology experts; and (2) reliability analysis, using the test-retest method, to assess the questionnaire’s internal consistency (Cronbach’s alpha) and reproducibility (intraclass correlation coefficient - ICC). The questionnaire includes 17 items assessing attitudes and knowledge about antibiotic prescribing and resistances and 9 items evaluating the importance of different sources of knowledge. The study was conducted in the catchment area covered by Portugal’s Northern Regional Health Administration and used a convenience sample of 61 primary-care and 50 hospital-care physicians. Results Response rate was 64 % (49 % to retest) for primary-care physicians and 66 % (60 % to retest) for hospital-care physicians. Content validity resulted in 9 changes to professional concepts. Face validity assessment resulted in 19 changes to linguistic and interpretative terms. In the case of the reliability analysis, the ICC values indicated a minimum of fair to good reproducibility (ICC > 0.4), and the Cronbach alpha values were satisfactory (α > 0.70). Conclusions The questionnaire developed is valid -in terms of face validity, content validity and reliability- for assessing physicians’ attitudes to and knowledge of antibiotic prescribing and resistance, in both hospital and primary-care settings, and could be a very useful tool for characterising physicians’ antibiotic-prescribing behaviour.