Background: Health professionals’ knowledge, beliefs and perceptions concerning radiation protection may affect their behaviour during surgery and consequently influence the quality of health services. This study highlights the health professionals’ average knowledge level and captures the beliefs, perceptions, and behaviours in a large public Greek hospital. Materials and Methods: A cross-sectional study was carried out, including health professionals working in operating rooms. One hundred thirty-two staff members participated by responding to an original questionnaire. The sample consisted of nurses, radiographers and medical doctors of various specialties involved daily in surgical procedures where ionizing radiation is required. The survey was conducted from March to June 2021, and the response rate was 97%. Results: The level of overall knowledge of health professionals regarding radiation protection safety was not satisfactory. Females and employees with a lower level of education had more misconceptions about radiation and radiation protection. Employees of younger ages and with less previous experience were more likely to have negative emotions towards radiation exposure. Finally, employees with fewer children tended to express physical complaints caused by their negative emotions due to radiation exposure. Conclusions: Health professionals’ lack of basic and specialized knowledge concerning radiation protection safety had a negative impact on the provision of health services. The continuing training of the staff seemed to be the only solution to reverse this trend. The training should highlight how radiation exposure can be minimized, safeguarding health professionals’ trust and sense of security by significantly improving their working environment.