Hand hygiene is a central component of patient care and infections prevention. This is a cost-effective preventive measure should be effectively used from advanced healthcare systems to primary healthcare units Objectives: To assess the practice of hand hygiene through a perspective observation among primary healthcare workers (PHCWs) with its correlation to their hand hygiene knowledge and to assess the efficacy of hand hygiene. Methods: This cross sectional study was conducted at primary care facilities at Kafer Shoker district, Qalyubia governorate, Egypt. WHO questionnaire for knowledge and observational check list of hand hygiene practice were used to assess the knowledge and practice of hand hygiene. A bacteriological study was conducted to swabs of the palms/fingers of the participants and were examined for microbial growths before and after perceived hand wash. Results: Among studied group, 82.6% of healthcare workers had moderate knowledge and 52.2% of them practiced hand hygiene appropriately. Factors as being female, nurse, increased experience duration, working in family medicine clinic, and previous hand hygiene (HH) training were associated with statistically significant practice of appropriate HH. The compliance rate to HH guidelines was appropriate only in 44.7 % of studied group. The microbiological results of this study showed significant efficacy of practiced hand washing (p < 0.001). Conclusion: There's a gap between self perceived practice and actual practice of HH.