Effective segregation practice of biomedical waste is a critical process in hospital waste management and maintenance of a ‘risk-free’ health care environment. Failure to appropriately identify and isolate infectious from non-infectious wastes could lead to a myriad of health and biomedical waste management issues, which include high risk of cross-contamination, high risk of infection, difficulty in the assessment of waste generation, categorization, and composition of hospital waste. The aim of this study is to assess the segregation practice among health workers in selected hospitals in Abuja towards achieving effective waste management. A descriptive and cross-sectional study was conducted among 430 participants from six hospitals in Abuja. Data were collected using a pretested, structured questionnaire and on-the-spot observation. Reliability analysis and descriptive statistics were employed for the analysis of data. Most of the respondents agreed to the segregation at the point of generation 366 (91.5%), majority of the respondents supported the use of color-coded bins for segregation. The Cronbach’s alpha α = 0.830, loading factor (0.601- 0.949) which indicates that the measuring tool was reliable and the items on the questionnaire showed co-relatedness and consistency. The study revealed that most of the respondents were sensitized and practiced the process of segregation. Although these practices were observed on the spot, they could later change thus, sustenance of these practices is expedient to maintain a lasting and workable waste management framework. Government and hospital waste management team to prioritize the segregation process through an adequate budget for supplies of segregation materials.