Background: Hand hygiene is effective in preventing and controlling healthcare-associated infections (HCAIs); however, its compliance remains low, and reasons are poorly understood, thus its research and feedback may lead to successful interventions. This study aimed to explore the observed and self-reported hand hygiene compliance and associated factors among healthcare workers (HCWs) in the medical/surgical ward of a county referral hospital. Methods: Cross-sectional study design was used in this study. Direct observation guided by 5 moments for hand hygiene concept was carried to describe compliance, and the questionnaire technique informed by Theoretical Domains Framework (TDF) was employed to investigate self-reported compliance, barriers, and facilitators of hand hygiene compliance. This study was done at a county referral hospital in Kenya. The total number of hand hygiene opportunities observed was 347 among 55 HCWs, and 132 HCWs filled the questionnaires. Results: The overall compliance based on direct observation was 23.9%, whereas self-reported compliance was 80.0%. Hand hygiene was significantly associated with indication (p < 0.001), training (p = 0.014), social influences (p = 0.002) and knowledge (p = 0.044). Barriers identified were environmental resources, social influences, beliefs about consequences, memory, attention, and decision processes. Facilitators included knowledge and social/professional role, and identity.Conclusions: Despite the high report rate of hand hygiene compliance, the observed compliance is unacceptably low. Although knowledge, and social/professional role and identity of the HCWs may primarily facilitate hand hygiene compliance, barriers associated with non-compliance such as environmental resources remain significant constraints and should be considered to ensure optimal hand hygiene compliance and quality of patient care. The results also suggest that HCWs education on hand hygiene still needs improvements, particularly on the 5 moments for hand hygiene.