Background: Hand hygiene compliance (HHC) can be influenced by behavioural determinants, but knowledge on this remains scarce. The Capability, Opportunity, Motivation-Behaviour (COM-B) hand hygiene questionnaire was developed by Lydon et al. to gain insight into self-reported behavioural determinants and self-reported HHC. Aims: To determine the validity of self-reported HHC using the COM-B questionnaire; and investigate the influence of self-reported behavioural determinants on observed HHC, taking environmental determinants into account. Methods: This was a cross-sectional study, from September to November 2019, in nine hospitals in the Netherlands. Healthcare workers (HCWs) completed the COM-B questionnaire, and direct hand hygiene observations were performed. In addition, information on environmental determinants (workload, ward category, hospital type and ward infrastructure) was collected. Validity of self-reported HHC was determined using the intraclass correlation coefficient (ICC). Univariable and multi-variable regression analyses were performed to investigate the relationship between behavioural and environmental determinants and observed HHC. Findings: The ICC showed no association between self-reported HHC and observed HHC [0.04, 95% CI -0.14 to 0.21]. In univariable regression analyses, ward category and the opportunity and motivation subscales were significantly associated with observed HHC. In multi-variable regression analysis, only ward category and the motivation subscale remained significant. Conclusion: Self-reported HHC is not a valid substitute for direct hand hygiene observations. Motivation (behavioural determinant) was significantly associated with HCC, while almost none of the environmental determinants had an effect on observed HHC. In further development of hand hygiene interventions, increasing the intrinsic motivation of HCWs should receive extra attention.