Abstract The objective of this study was to assess the level of knowledge, protection and awareness of radiation among healthcare professionals. A total of 413 healthcare professionals from two major training and research hospitals in Istanbul took part in the study. The sample consisted of 26.6% physicians and 73.4% other healthcare professionals. There was considerable variation in the knowledge of radiation content in imaging techniques, with correct response rates of 89.3% for Computed Tomography, 80.1% for digital x-ray, and 52.8% for Magnetic Resonance Imaging. While 69.5% of respondents provided correct answers to questions regarding radiation protection principles, 64.4% demonstrated a lack of sufficient knowledge in this area. It is noteworthy that 48.4% of respondents incorrectly identified the medical imaging technique to which the public is most exposed. It was concluded that further training on radiation-related issues should be provided at the university level and through in-service training for healthcare professionals.