Hand hygiene is among the most important factors of infection control in healthcare settings. Healthcare workers are 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). The 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 < 0.05). Private healthcare workers were less likely (OR: 0.33, p < 0.01) to have moderate/good perceptions compared to the government healthcare workers. Healthcare workers who received training on hand hygiene were more likely to have good/moderate perception (OR: 3.2, p < 0.05) and to routinely use alcohol-based hand rubs (OR: 3.8, p < 0.05) than the ones without such training. Physicians are more likely (OR: 4.9, p < 0.05) to routinely use alcohol-based hand rubs than technicians. Our research highlighted gaps in hand hygiene knowledge, perception and practice among healthcare workers in Qassim, Saudi Arabia and the importance of training in this regard.