This paper examined the knowledge, compliance and impact of hand hygiene among healthcare professionals during COVID-19 outbreak in South-East, Nigeria. The data used in this study were collected from twenty (20) hospitals in South-East, Nigeria using questionnaire with closed-type question forms. A total number of 600 questionnaires were used in this study. Two-way CATANOVA was used to examine the gender and health profession that have well knowledge, compliance and impact experience of hand hygiene during COVID-19 outbreak. The result showed no statistically significant difference in the knowledge, compliance and impact experience of hand hygiene among four major health professions (medical doctors, nurses, pharmacists, laboratory scientists) and also between the genders at a 5% significance level. The findings showed that the changing of healthcare professional from one health profession to another does not affect the knowledge, compliance and impact experience of hand hygiene. It was noticed that 599(99.8%) healthcare professionals have good knowledge of hand hygiene, 395(65.8%) practice hand hygiene every time, and 507(84.5%) have high impact experience of hand hygiene. There is enhancement in the knowledge, compliance and impact experience of hand hygiene of healthcare professionals as their years of service increase.