Healthcare-Associated Infections (HAIs) remain a major problem in hospitals as they contribute to increased morbidity, mortality, length of hospital stay, and healthcare costs. One of the most effective measures to prevent HAIs is the implementation of the Five Moments of Hand Hygiene. Nurses’ compliance with this practice is assumed to be related to their level of knowledge. This study aimed to determine the relationship between nurses’ knowledge level and compliance with the implementation of the Five Moments of Hand Hygiene in the inpatient wards of Karangasem Regional General Hospital.This study employed a quantitative design with a cross-sectional approach. The population and sample consisted of all staff nurses working in the inpatient wards of Karangasem Regional General Hospital, totaling 125 respondents, using a total sampling technique. Data on knowledge level were collected using a questionnaire, while hand hygiene compliance was measured using an observation checklist. Data analysis was conducted using univariate and bivariate analyses with the Spearman Rank correlation test at a significance level of 0.05. The results showed that most nurses had a good level of knowledge, with 108 respondents (86.4%). Nurses’ compliance with the implementation of the Five Moments of Hand Hygiene was also predominantly in the good category, with 102 respondents (94.4%). Bivariate analysis revealed a significant relationship between nurses’ knowledge level and compliance with the Five Moments of Hand Hygiene at Karangasem Regional General Hospital (p < 0.05). It can be concluded that there is a significant relationship between nurses’ knowledge level and compliance with the implementation of the Five Moments of Hand Hygiene. Improving nurses’ knowledge through continuous education and training is expected to enhance compliance and support efforts to prevent HAIs in the hospital setting.