Background: Segregation of biomedical waste (BMW) is the foremost and prime step for effective BMW management. Inadequate knowledge or a lower level of skills among nursing staff in healthcare facilities has led to the failure of BMW management. Therefore, they should have competencies in knowledge, performance, and attitudes about BMW segregation in healthcare settings. Aims:The current study aimed to assess nursing staff's knowledge, performance, and attitude about BMW segregation. Design: A descriptive cross-sectional study design was used. Setting:The study was carried out at Mansoura Health Insurance Hospital, affiliated with the Ministry of Health and Population. Sample: In a convenient sample, all on-the-job nursing staff (300) were included Tools for data collection: A structured, self-administered questionnaire scale and an observation checklist were used for data collection. Results: The results showed that 64.3%, 100%, and 83.3% of the nursing staff had poor total knowledge, improper performance, and a positive attitude toward score levels, respectively, regarding BMW segregation. Conclusion: The study concluded that almost two-thirds, all of them as well as the majority, of the nursing staff have poor knowledge, improper performance, and positive attitudes, respectively, related to BMW segregation. Recommendations: The researchers recommend conducting on-the-job training programs for nursing staff on BMW segregation measures.