Religious, economic, political, social, and cultural factors influence professional autonomy in nursing, and differ from country to country. The aim of this study was to explain barriers and facilitators of professional autonomy in the experience of clinical nurses. This qualitative study was conducted on 19 clinical nurses and nursing managers selected based on purposive sampling from January 2018 to December 2019. Data were collected through semi-structured interviews, which continued until data saturation was reached. Conventional content analysis based on the Graneheim and Lundman approach was used to analyse the data. Data were categorised into 34 subcategories, 8 main categories, and two main themes, which were titled as facilitators of professional autonomy (professional, organisational, and individual factors, and effective communication) and barriers to professional autonomy (professional, organisational, and personal barriers, and inappropriate communications). The results discussed a set of facilitators and barriers faced by Iranian nurses in clinical settings. Professional organisations can play a key role by enhancing professional autonomy facilitators and increasing professional support for nurses. Another finding was the importance of training nursing staff to be competent and autonomous, beginning right from the college years.