Introduction: Intraosseous vascular access involves the insertion of a specialized hollow needle through the bone cortex into the medullary space for the administration of medical therapy and laboratory testing. This technique serves as an alternative when standard venous access is unavailable, thus preventing delays in the urgent delivery of treatment. The success rates of intraosseous access are twice as high as the success rates for establishing an intravenous line in patients with critical trauma and no blood pressure, making intraosseous access preferable in such situations. Objective: The aim of this thesis is to assess the level of knowledge among nurses and technicians in the Emergency Medical Service regarding intraosseous access. Additionally, based on the obtained results, the goal is to determine whether there are differences in the level of knowledge among nurses and technicians with respect to their level of education and length of work experience. Participants and Methods: The research was conducted through a questionnaire on a convenience sample of 84 employees of the Emergency Medical Service of the Republic of Croatia (N=84). An online questionnaire was used, consisting of two parts: the first part collected sociodemographic data, while the second part contained a knowledge test with 8 multiple-choice questions. Results: The research showed that 52.4% of the respondents have good knowledge of intraosseous access, with a median of 5 correct answers (IQR 3–6). Statistically significant differences in knowledge levels were observed based on gender (p = 0.045), with men showing better results. Significant differences were also observed based on the level of education (p = 0.020), with respondents with higher education achieving better results. No statistically significant difference in knowledge level was observed with respect to the length of work experience (p = 0.316). Conclusion: The research indicates that the level of education significantly influences the level of knowledge among nurses and technicians about intraosseous access, while the length of work experience is not associated with higher knowledge levels. The results suggest a need for additional education to ensure quality healthcare and to increase the knowledge level regarding this important medical technique.