Background: Of the estimated 384,000 needle-stick injuries occurring in hospitals each year, 23% occur in surgical settings. This study was conducted to assess safe injection procedures, injection practices, and circumstances contributing to needlestick and sharps injures (NSSIs) in operating rooms. Methods: A descriptive cross sectional approach was adopted. Modified observational checklists based on World Health Organization (WHO) definitions were used in operating rooms (n = 34) and interview questionnaire was administered to HCWs (n = 318) at the Alexandria Main University Hospital. Results: Safe injection procedures regarding final waste disposal were sufficiently adopted, while measures regarding disposable injection equipment, waste containers, hand hygiene, as well as injection practices were inadequately carried out. Lack of job aid posters that promote safe injection and safe disposal of injection equipment (100%), overflowing of sharps containers and presence of infectious waste outside containers (50%), HCWs not cleaning their hands with soap and water or alcohol-based hand rub (58.1%), and HCWs not wearing gloves during IV cannula insertion (58.1%), were all findings during observations. High prevalence of NSSIs was reported (61.3%), mostly during handling suture needles (50.8%). In addition, 66.2% of the injured HCWs were the original user of the sharp item which was contaminated in 80% of injuries. At time of NSSI, 79% HCWs were wearing gloves. The most common injured sites were left fingers (39.5%), and 55.4% of injuries were superficial. After exposure, 97.9% did not report their exposure. The source patient was not tested for HBV, HCV and HIV infection in more than 70% of injuries and 96.9% of injured HCWs did not receive post exposure prophylaxis. Conclusion: The study highlighted that inadequately adopted safe injection procedures and insufficient injection practices lead to high prevalence of NSSIs in operating rooms.