Introduction: Sterilization is one of the basic principles and techniques in the systematic fight against infections. The sterilization methods depend primarily on the material of which the articles are made and the instructions given by the manufacturers of the material to be sterilized. The nurse/technician, as well as other staff involved in sterilization work, must first and foremost be high quality and professionally trained for the work they do. In addition to education, they should be fast, agile, responsible, conscientious, and aware of the difficulty and responsibility of the work they do. Often, healthcare professionals are unfamiliar with the job and responsibilities of staff at the Central Sterilization Unit. Aim: The aim of this paper was to determine attitudes and knowledge about sterilization of medical and consumables among health professionals at the Clinical Hospital Center Zagreb, and to determine whether there is a correlation between the level of education with attitudes and knowledge and whether the workplace influences attitudes and knowledge about sterilization of medical and consumables. Methods: The study involved 240 subjects, conducted between March and June 2019. Included were health professionals using the services of the Sterilization Division. An anonymous survey questionnaire was prepared as an instrument of research, which was created for the purpose of this research (designed by the author of the paper), the implementation of which received ethical approval from the Clinical Hospital Center Zagreb and was not used for other purposes. Results: There were no significant gender differences in attitudes and knowledge about sterilization of medical and consumables among the respondents. Respondents working in Operations Departments showed more knowledge than respondents employed in non-Operations Departments. In addition to knowledge, there is a statistically significant difference in attitudes among the observed groups. A significant difference was found with regard to the respondents′ professional qualifications only with regard to attitudes. There is a significant difference in attitudes about the sterilization of medical and consumables among the employees employed in the surgical wards versus those from other non-surgical wards. Conclusion: The results obtained show that there is no statistically significant gender difference in attitudes and knowledge, while the workplace influences attitudes and knowledge about sterilization of medical and consumables. The statistical analysis of the data obtained shows that education influences attitudes, but not the knowledge about sterilization of medical and consumables.