This study aimed to evaluate medical students’ hand hygiene knowledge at Balqa Applied University. A cross-sectional study was conducted using the World Health Organization Hand Hygiene Knowledge Questionnaire for Health-Care Workers. Knowledge scores were calculated based on correct responses, and independent-samples t-tests were used to examine differences in knowledge scores between groups. A total of 292 medical students participated. The mean knowledge score was 14.5 ± 2.7 out of 25. Most students demonstrated moderate knowledge (70.9%), while 24.0% had poor knowledge and 5.5% had good knowledge. Knowledge scores did not differ significantly by sex, medical year, or routine use of alcohol-based handrub (p > 0.05). However, students who had received formal hand hygiene training or education achieved significantly higher scores (p = 0.023). Overall, medical students demonstrated a moderate level of hand hygiene knowledge, and formal training or education was associated with better knowledge, emphasizing the value of incorporating hand hygiene education and training into medical curricula.