Background: Hand hygiene is widely recognized as a simple way to prevent infection transmission to patients or among health care providers and adherence to its measures is associated with a significant reduction in mortality and morbidity. Despite this, many studies conclude that hand hygiene compliance is suboptimal. This study aims to assess compliance of healthcare workers to hand hygiene measures in Khartoum oncology hospital. Method: A cross-sectional hospital-based descriptive study was conducted during the period from December 2021 to July 2022 on a sample of 109 health care workers [doctors and nurses]. Hand hygiene knowledge and practice of health care workers were measured using a structured, pretested, administered questionnaire, modified from the WHO standardized questionnaire for HH knowledge and observation technique with a checklist was used to collect data on the practice. The data is analyzed by frequency tables. The Chi-square test is used to determine associations among categorized variables. All statistical tests were considered significant when the p value was 0.005. Result: Of the 109 participants, 56% had not received formal hand hygiene training in the last three years. 18.3% did not use alcohol for hand hygiene in their practice. Regarding participants' knowledge of the importance of hand hygiene, 97.2% considered it an important tool for infection prevention. Practice wise, 60% of participants did not wash their hands before touching the patients, while 20% of them used the same glove for more than one patient. A significant statistical association was found between the routine use of alcohol and receiving formal training in hand hygiene in the last three years (p-value 0.004). Conclusion: The overall use of alcohol-based hand rub for hand hygiene was high, in spite of low training programs in the last three years. There is a huge shortage of soap and alcohol in working places. Developing a training program will help magnify the use of alcohol for hand hygiene.