Objectives: Although the World Health Organization (WHO) initiative “My 5 Moments for Hand Hygiene” has been lauded as effective in preventing hospital-associated infections, little is known about healthcare workers (HCWs)’ hand hygiene behavior. In this study, we sought to assess knowledge and attitudes towards the concepts in this initiative, as well as associated factors, among Vietnamese HCWs at a general hospital. Methods: A structured questionnaire was administered to HCWs at a central Vietnamese general hospital in 2015. Multiple logistic regression analysis was used to identify factors associated with HCWs’ knowledge and attitudes towards hand hygiene. Results: Of 120 respondents, 65.8% and 67.5% demonstrated appropriate knowledge and a positive attitude, respectively, regarding all 5 hand hygiene moments. Logistic regression indicated better knowledge of hand hygiene in workers who were over 30 years old, who were direct HCWs (rather than managers), who had frequent access to clinical information, and who received their clinical information from training. Those who worked in infectious and tropical disease wards, who had frequent access to clinical information, and who received information from training were more likely to have a positive attitude towards hand hygiene than their counterparts. Conclusions Although many Vietnamese HCWs displayed moderate knowledge and positive attitudes towards the WHO hand hygiene guidelines, a key gap remained. Regular education and training programs are needed to increase knowledge and to improve attitudes and practices towards hand hygiene. Furthermore, a combination of multimodal strategies and locally-adapted interventions is needed for sustainable hand hygiene adherence.