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The accuracy of direct hand hygiene observation is challenged by Hawthorne effect.Hawthorne effect was quantified at a tertiary care setting in the current study.Overall hand hygiene compliance was 87.1% using overt observation.Overall hand hygiene compliance was 44.9% using covert observation.The overestimation was seen in all professions, hospital settings, and indications.Although direct human observation of hand hygiene (HH) is considered the gold standard for measuring HH compliance, its accuracy is challenged by the Hawthorne effect.To compare HH compliance using both overt and covert methods of direct observation in different professional categories, hospital settings, and HH indications.A cross-sectional study was conducted in 28 units at King Abdulaziz Medical City, Riyadh, Saudi Arabia, between October 2012 and July 2013. Compliance was defined as performing handrubbing or handwashing during 1 of the World Health Organization 5 Moments for HH indications (ie, opportunities). Overt observation was done by infection preventionists (IPs) who were doing their routine HH observation. Covert observation was done by unrecognized temporarily hired professionally trained observers.A total of 15,883 opportunities were observed using overt observation and 7,040 opportunities were observed using covert observation. Overall HH compliance was 87.1% versus 44.9% using overt/covert observations, respectively (risk ratio, 1.94; P < .001). The significant overestimation was seen across all professional categories, hospital settings, and HH indications.There is a considerable difference in HH compliance being observed overtly and covertly in all categories. More work is required to improve the methodology of direct observation to minimize the influence of the Hawthorne effect.