Understanding antimicrobial prescribing attitudes and perceptions, especially in the early stages of medical training, is an important driver for appropriate interventions. This study examined junior doctors' knowledge, attitudes and perceptions regarding antimicrobial use and antimicrobial resistance in Greece. A self-administered, internet-based questionnaire survey was completed by trainees and residents of all primary care centres and hospitals in Crete, Greece. From the 313 invited junior doctors, 214 (68.4%) fully completed the questionnaire. The mean correct knowledge score (four items) was 60%. The highest confidence rates with prescribing were recorded in accurately diagnosing an infection that needs antimicrobial treatment, selecting an appropriate administration route, and selecting the appropriate dosage. The lowest confidence rates were recorded in modifying antimicrobial treatment based on clinical and/or microbiological evidence, prescribing antimicrobial combinations and not prescribing an antimicrobial in a febrile patient without severity criteria and uncertain diagnosis of infection. Use of guidelines and expert consultation were perceived as the most important factors influencing antimicrobial prescribing practices. Most participants were aware of the antimicrobial resistance problem in Greece, and considered excessive prescribing of antimicrobials and broad-spectrum antimicrobials to be the most important causes. Availability of local and national guidelines and antimicrobial resistance data, senior consultation and infectious diseases specialist consultation were the preferred interventions to improve antimicrobial prescribing. The findings of this study will contribute to the design of appropriate interventions, based on local evidence, for the establishment of antimicrobial stewardship programs in a country characterised by excessive use of antimicrobials and high resistance rates. Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.