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The current prevalence of healthcare-associated infections (HCAIs) is a major public health concern. Patient contact in the operating theatre (OT) can contribute to HCAI via microbial contamination. The application of hand hygiene is effective in reducing infection rates. Limited data are available on adherence to hand-hygiene guidelines by OT staff.Covert direct observations of OT staff at an academic medical centre were performed by a single, trained observer. The primary outcome was the frequency of hand-hygiene application by OT staff, including anaesthesiologists, anaesthesia nurses, surgeons, surgical nurses, and medical students. ‘Sterile' scrubbed staff members were excluded. The following hand-hygiene opportunities were monitored: (i) entering or leaving the OT; and (ii) before patient contact. Furthermore, the frequency of ‘potential contamination' was recorded (touching OT implements after contact with patient/patient body fluids without the subsequent application of hand hygiene). We recorded non-surgical glove usage for invasive procedures, for example, intubation or insertion of intravascular devices. Finally, we collected qualitative data on incentives for hand hygiene.A total of 28 operations were observed (60 h of observations). On average, 0.14 hand-hygiene applications per hour per staff member were witnessed. Upon entering or leaving the OT, hand hygiene was performed in 2% (7/363) and 8% (28/333) of opportunities.Frequent interactions between patient, staff, and OT environment were observed. Adherence to hand-hygiene guidelines by OT staff was extremely low. This potentially exposes patients to microbial transmission, HCAIs, and patient harm.