Based on recommendations from World Health Organization, hand hygiene is the most important way to control the hospital infections. Due to the critical role of nurses in patient care, they should have essential and updated information regarding hand hygiene. So this study aims at determining the knowledge of hand hygiene and its individual and organizational predictors among nurses in neonatal units. This descriptive and cross-sectional study was conducted in neonatal units in the hospitals affiliated to Tabriz University of Medical Sciences. The participants surveyed in this study were 150 nurses who were invited by census sampling method. A researcher prepared questionnaire that investigated the knowledge of participants about hand hygiene and was used after approving its validity and reliability. The quantitative analysis of this study used Statistical Package for Social Sciences SPSS version 13 by descriptive statistics and pearson correlation test, independent samples t-test, One-way ANOVA. For multivariable explanation of nurses' knowledge based on independent variables multiple linear regressions was used. Most of participants have an acceptable level of knowledge regarding hand hygiene. The highest score was for infection control domain and the lowest score was for definition of hand hygiene domain. Multivariable analysis showed that work experience and history of previous training were the most important predictors of participants' knowledge about hand hygiene. It is recommended that infection control committees should revise their educational methods and give more emphasis on update guidelines regarding hand hygiene. Also, more experienced nurses should be employed in neonatal units.