Background: Healthcare-associated infections (HAIs) are a significant concern in healthcare services due to their association with extended hospital stays and the potential for morbidity or fatality. Ensuring proper hand hygiene (HH) is the foremost measure in HAIs. The objective was to evaluate HH perception, knowledge, and compliance among healthcare workers (HCWs), at the National Liver Institute (NLI), Menoufia University. Methods: In this descriptive cross-sectional work, 100 HCWs at NLI Menoufia University departments were investigated using standardized World Health Organization's (WHO) knowledge and perception questionnaires. HH compliance was evaluated using the WHO Five Moments HH Observation form as the reference. Results: Of 100 HCWs at NLI (35 doctors, 65 nurses), half of the HCWs were females (51%) aged between 21 and 35 years. The overall knowledge level showed that only 16% of studied HCWs had a good level of HH-related knowledge and over half of them (58%) had a moderate level while 26% of the HCWs had poor knowledge. Regarding perception survey, more than half of the HCWs (53%) had moderate perception, 32% of HCWs had good perception, and only 15% of HCWs had poor perception. The overall HH compliance was 38.1% out of 672 total HH opportunities. Nurses had a higher compliance rate than doctors (43.5% vs. 24.5% respectively). Conclusions: The study highlights notable disparities in HH knowledge and practices, with nurses demonstrating better awareness and adherence compared to doctors. Despite the high perception of HH's importance, overall compliance was suboptimal, particularly in certain departments and moments recommended by WHO.