•A framework for assessing nuclear security culture was introduced.•A survey on the level of nuclear security culture at various radiological facilities was conducted.•Factors considered in the analysis: age, qualification, job description and sector of radiological practice.•The perception of nuclear security culture was found generally increasing with age.•Staff of higher education were found more capable of forming their own opinions in security matters. Experience proves that nuclear security culture is crucial to effectively securing radioactive and nuclear materials from external and internal adversaries. There are some approaches to assess the effectiveness of nuclear security culture in a certain organization, such as conducting surveys, interviews and onsite observations. This paper introduces a framework for effective assessment of nuclear security culture and evaluates the level of security awareness among workers at various radiological facilities. The proposed framework comprises four elements that are: self-assessment, nuclear security culture indicators, measurement and analysis, and communication of results. A survey, to assess the degree of perception of security culture at various facilities using radioactive materials, was developed. The survey was based on an online questionnaire of 32 questions, grouped in six categories which are: Beliefs, Principles, Values, Characteristics, Attitudes and Behaviors. Information regarding awareness of nuclear security culture was gathered and analyzed, in addition to information related to respondents that included: age, qualification, job description and sector of radiological practice, which are: medical, industrial, research and regulatory sectors. The data from the collected responses has revealed information about the degree at which the respondents believe that security threats are real concern for their facilities and the level of readiness to contribute to security matters at their organizations. It also shows trends on the degree of understanding personal responsibilities to protect the facility from various threats. It also demonstrates the extent into which the respondents think that the security community values the contribution that scientists, engineers and other radiation users can make to nuclear security effectiveness and the degree of belief that scientists and engineers are actively engaging in security matters. The results of the survey have revealed that the perception of nuclear security culture generally increases with age. Results have also shown that employees with managerial responsibilities are slightly more appreciating the security measures implemented at their organizations. The results also demonstrated that the staff with higher level of education are more likely to be of higher critical sense and more capable of forming their own opinions in security issues and sharing them with others.