Background: Physical activity (PA) is hypothesized to reduce the risk of several chronic diseases and enhance longevity. Low levels of activity have been associated with some but not all types of cancers. We expected that work experience at National Cancer Institute (NCI) might have a positive effect on knowledge about and practice of physical activity among its workers. Aim: To describe physical activity practice among workers (medical & nonmedical) in National Cancer Institute (NCI), Cairo University and to measure knowledge about different health benefits of physical activity and its role in cancer prevention and treatment. Participants & method: This is a cross-sectional survey with anonymous self-administered questionnaire for workers (medical and non-medical). Their level and pattern of physical activity were assessed using modified International Physical Activity Questionnaire version 8, their knowledge about health benefits of PA and its relation to cancer prevention and treatment were studied. The PA level was estimated as metabolic equivalent task (MET minutes/week). Results: The medical group of workers was significantly more knowledgeable about different health benefits of regular physical activity (PA), however the whole group were lacking adequate knowledge about role of PA in cancer prevention and treatment. No significant difference was found between the medical and non medical workers as regard the activity level. The most common barrier to participate in regular PA among the whole studied group was insufficient time. Significant variables affecting total score MET minute were age groups, work type, job hours per week, education level (years), presence or absence of children, sitting minutes/day. Only 23.5% of the whole studied groups participate in recreation and sport PA. Conclusion: There was lack of adequate knowledge about relation between PA and cancer prevention and its role in cancer treatment. Recommendations: Development and implementation of a health-related physical education program about benefits of PA practice. Additionally, suitable place and equipments should be made available at the NCI to stimulate workers for PA participation.