Hand hygiene is among the most important factors of infection control in healthcare settings. Healthcare workers are considered the primary source of hospital acquired infection. We assessed the current state of hand hygiene knowledge, perception and practice among the healthcare workers in Qassim, Saudi Arabia. In this cross-sectional study we used the hand hygiene knowledge and perception questionnaire developed by the World Health Organization. Knowledge and perceptions were classified into good (80 – 100%), moderate (60 – 79%) and poor (<60% score). Majority of the healthcare workers had moderate knowledge (57.8%) and perception (73.4%) of hand hygiene. Males were less likely to have moderate/good knowledge compared to females (OR: 0.52, p<.05). Private healthcare workers were less likely (OR: 0.33, p<0.01) to have moderate/good perception compared to the government healthcare workers. Healthcare workers who received training on hand hygiene were 3.2 times likely (p<.05) to have good/moderate perception and 3.8 times likely (p<0.05) to routinely use alcohol-based hand-rub than the ones without such training. Physicians were 4.9 times likely (p<0.05) to routinely use alcohol-based hand-rub than the technicians. Our research highlighted gaps on hand hygiene knowledge and perception and practice among healthcare workers in Qassim and importance of training in this regard.