Hand hygiene (HH) is vital for preventing healthcare-associated infections and ensuring patient safety. This study evaluated the knowledge, beliefs, and practices of healthcare professionals in primary healthcare centers (PHCs) in Riyadh, Saudi Arabia. A cross-sectional descriptive study was conducted using an online validated questionnaire based on the World Health Organization Hand Hygiene Knowledge Questionnaire. Data were analyzed to identify gaps and patterns across professional roles and demographics. A total of 221 healthcare professionals participated, 76% women and 24% men, 57% aged 30–39 years. Most had over 15 years of experience (33%), with nurses comprising the largest professional group. Nearly all participants (91%) had received HH training in the past 3 years, and 88% reported routine use of alcohol-based hand sanitizer. However, only 67% correctly identified the minimum time for alcohol-based sanitizer to kill germs, and misconceptions about hand rubbing efficacy persisted. The primary route of germ transmission was identified as healthcare workers’ unclean hands. Most participants agreed that hand rubbing is faster than handwashing and emphasized HH after patient contact, exposure to body fluids, or contact with the patient’s surroundings. Nearly all endorsed using both hand rubbing and handwashing for specific scenarios, such as before injections. This study highlights strong HH knowledge and adherence among healthcare workers in Riyadh PHCs, reflecting effective training programs. However, persistent misconceptions and knowledge gaps regarding germ transmission and hand rubbing efficacy require targeted interventions.