: Hand hygiene is the most effective recommended practice to prevent hospital-associated infections. Although, hand hygiene compliance rate is still low. The general purpose of this study was to describe Jordanian nurses' knowledge, attitudes, and barriers toward hand hygiene. A non-experimental descriptive cross-sectional design was used for this study. Validated self-reported questionnaires were used among 207 nurses (106 male and 101females) from four major hospitals in Jordan. Nurses have a moderate level of knowledge (13.83±2.64) and moderate positive attitudes (32.74±4.54). Furthermore, nurses who were working in private hospitals had higher levels of knowledge than nurses who work in teaching hospitals who had higher levels of knowledge than nurses working in governmental hospitals. Nurses who were working in private hospitals had higher levels of attitude than nurses working in teaching and governmental hospitals. Surgical nurses had higher levels of an attitude than ICU nurses and higher levels than medical nurses. On the other hand, ER nurses had shown the lowest attitude. In addition, the result showed that the most important barriers toward hand hygiene were: 1) insufficient time, 2) skin irritation, 3) wearing gloves, 4) lack of knowledge, and 5) ignorance of guidelines. In conclusion, there is a need for continuous education about hand hygiene in order to improve hand hygiene knowledge and to enhance the change in attitude. This study revealed that some of the major barriers were workload, skin irritation and wearing gloves. Management should reduce workload and provide appropriate hand hygiene products.