Securing radioactive sources has become increasingly important given the rising threat of radiological terrorism. While radiation safety has long been established in most applicable industries, the importance of nuclear and radiological source security has lagged behind in nonnuclear material specific industries, such as academic institutions and medical facilities. To evaluate the attitudes and behaviors regarding nuclear security culture, an assessment of nuclear and radiological material practices was developed and conducted on 73 radioactive material users at a university. The survey portion of the assessment was comprised of a series of questions segregated into four categories: policy, enforcement, leadership, and behavior. Nuclear security awareness questions formed a subset of the questionnaire. Users were classified by their radioactive material experience and work classification: student, faculty, or other staff. Of the users surveyed, 9% were also interviewed face-to-face to further expand on their views of nuclear security culture. Results of the assessment showed that students from the work classification group as well as the cohort of radioactive material users with 2–5 y of experience possessed a greater degree of awareness towards nuclear security compared to faculty and other more experienced radioactive material users. Relative to students and faculty, other staff from the work classification group faced some difficulty judging the enforcement of policies, adequacy of inspection, and job performance review related to nuclear security. The response from all three groups emphasized the need to enhance threat-response preparedness and greater communication among stakeholders.