Background.In 2009, the World Health Organization (WHO) recommended “My Five Moments for Hand Hygiene” (5MHH) to optimize hand hygiene (HH). Uptake of these recommendations by healthcare workers (HCWs) remains uncertain.Methods.We prospectively observed HCW compliance to 5MHH. After observations, eligible HCWs who consented to interviews completed surveys on factors associated with HH compliance based on constructs from the transtheoretical model of behavioral change (TTM) and the theory of planned behavior (TPB). Survey results were compared with observed HCW behaviors.Results.There were 968 observations among 123 HCWs, of whom 110 (89.4%) were female and 63 (51.3%) were nurses. The mean HH compliance for all 5MHH was 23.2% (95% confidence interval [CI], 18.1%-28.3%) by direct observation versus 82.4% (95% CI, 79.9%-84.9%) by self report. The HCW 5MHH compliance was associated with critical care unit encounters (P < .05), medicine unit encounters (P = 0.08, P <.001), immunocompromised patient encounters (P <. 05), and HCW prioritized patient advocacy (P <.001). Self-reported TTM stages of action or maintenance (P = .08) and the total TPB behavior score correlated with observed 5MHH (r = 0.21, P = .02) and with self-reported 5MHH compliance (r = 0.53, P < .001).Conclusion.Observed HCW compliance to 5MHH was associated with the type of hospital unit, type of provider-patient encounter, and theory-based behavioral measures of 5MHH commitment.