Background: Ensuring safe injection practice is one of the greatest challenges for healthcare system in developing countries. Objectives: to assess the injection safety practices by evaluating knowledge and practices of HealthCare Workers (HCWs) towards injection safety before and after training program, and to determine the incidence of needle stick injuries (NSIs) among HCWs. Methodology: A cross-sectional study was conducted at Fayoum University Hospitals from October 2016 to June 2017. Two hundreds HCWs working in eighteen departments were included. Questionnaire was used to collect information about knowledge, and 395 injection opportunities were observed using a standardized observation check list to detect practices of HCWs towards injection safety before and after training program. Results: Significant change in knowledge of HCWs was detected between pre and post training intervention (p <0.05). Regarding the practices, significant improvement nearly in all items of observation checklist (p<0.05) were observed. The incidence rate of NSI was 27/200 (13.5%).The best knowledge and practices mean percentage was obtained from neonatology staff (p< 0.001), and the poor knowledge (p< 0.04) and practices (p< 0.02) mean percentage were from internal medicine staff either pre or post training. Conclusion: Educational Intervention found to be effective in improvement of safety injection practices.