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This study aims to assess hand hygiene compliance and associated factors among healthcare providers in Dessie referral hospital (Ethiopia) using the gold standard WHO's observational checklist and self-administered questionnaire. Hand hygiene practices of 230 healthcare providers from ten hospital wards were observed over 24 hours period. The required numbers of sample were proportionally allocated among the different professions and wards. The overall observed hand hygiene compliance was only 17.6%. Hand hygiene training , availability of functional sink , knowledge about hand hygiene , attitude towards hand hygiene , availability of water and soap , and availability of alcohol-based hand rub were positively associated with hand hygiene compliance. As lower levels of compliance were observed for indications that have potential risk for patients, healthcare providers need to follow the WHO's multimodal hand hygiene improvement strategies. This study also indicated the important prerequisites that could help improve hand hygiene.