Background: Hand hygiene is the simplest and most cost-effective practice for controlling health care associated infections (HCAIs). Level of adherence and determinants of poor compliance to hand hygiene (HH) among health care workers (HCWs) should be investigated in all health care settings. Objective(s): The study was conducted to assess knowledge and attitudes of health care workers towards hand hygiene and to assess their degree of compliance with moment one hand hygiene. Methods: A cross sectional study including HCWs (residents and nurses) in Alexandria Main University Hospital was conducted. The World Health Organization questionnaires and observational checklists were used to collect data in addition to a structured questionnaire to assess attitudes towards hand hygiene. Knowledge and attitude scores were calculated. Results: Only 15.5% of the studied HCWs had satisfactory level of hand hygiene related knowledge and more that half of them (55.8%) had a fair level of knowledge. The majority of HCWs (91.3%) agreed that hand hygiene practices are not practical in emergency situations. About half (49.5%) of them didn't feel guilty when they omit hand hygiene practices. Hand hygiene practices were missed in the vast majority of opportunities (>95%). Gloves were not available at any of the internal medicine or surgical wards but were rarely or intermittently available at the studied intensive care units (66.7% and 33.3% respectively). The most common cited barriers to hand hygiene practices among the studied HCWs were lack of sinks, soap, paper towels and alcohol-based hand rub. Conclusion: Training, education and motivation of HCWs in Alexandria Main University Hospital in addition to availability of the required resources and supportive environment are the best ways to improve the level of compliance with hand hygiene.