Needlestick injury remains the potential source for transmission of blood born infection and one of the main safety concerns which need to be addressed for the prevention of various blood-borne diseases among HCWs. The aim of our study was to assess the knowledge and practice gaps about Needle Stick Injuries (NSIs) and their associated factors among Health Care Workers’ (HCWs) at Omdurman teaching hospital. Methods: A cross-sectional study was conducted in may–June2016. (92) Respondents enrolled in the study (medical officer, lab technicians and nurses selected randomly. Results: Overall the knowledge about transmission of HBV HCV and HIV was good, mean knowledge 76.1%, in general, their practice regard news was poor means score practice 88.9%.there is significant relation between knowledge and socio-demographic data p .000 and insignificant between socio-demographic and their practice p.612 From the study concluded that NSSIs are common risk factors for infection among health care workers due to their poor practice, although their knowledge is good. Lastly recommended that Hospitals and healthcare facilities should be provided all preventive skills and instruments against infections. Record keeping and reporting of sharp injuries should be considered.