A nursing job is closely associated with a risk of exposure to a number of harmful factors. Every workplace has its own distinctive features which may result in a specific kind of occupational exposure. An operating theatre is a highly technical place, where irradiation constitutes one of the risks. Fluoroscopy is often used in surgery, because of technological development, which brings not only benefits, but also an extraordinary risk of medical staff being exposed to radiation. Therefore, this study aimed to evaluate operating theatre nurses’ knowledge on ionizing radiation and the principles of protection against radiation. A diagnostic survey was used as a method of analysis, in which an original questionnaire was applied as an expertise tool. The study involved 45 nurses employed in an operating theatre. The results indicate that the knowledge of the nurses on radiological protection is at a medium level. Moreover, it turned out that there is a trend indicating that with the length of service in the operating theatre not only does the level of knowledge in the field of radiological protection increase, but more importantly, it has impact on the nurses’ experience at complying with common principles of protection against radiation and at using personal protective equipment.