Transmission of pathogens from the hands of health care workers (HCWs) is the main cause of nosocomial infections, and hand hygiene is the single most important procedure to prevent it. At present, little is known about the adherence of HCWs to hand hygiene procedures in hematology units, where the patients are at high risk for acquiring hospital infections. In a prospective observational study, two observers monitored the hand hygiene compliance of HCWs in a hematology unit during 30-min observation periods distributed randomly during the daytime over 2 months. The prevalence of compliance with hand hygiene was 26% on 638 observed occasions. The noncompliance was higher among nurses [odds ratio (OR) 3.52, 95% confidence interval (CI) 1.85–6.70] and other HCWs (OR 1.72, 95% CI 0.98–3.02) compared to physicians. The compliance rate differed from 4 to 60% depending on the activity. The lowest compliance rate (4%) was observed before patient care and the highest (60%) was after insertion of invasive devices. When we classified the occasions for hand hygiene into ‘before’ and ‘after’ activities, the compliance rates were 9 and 36%, respectively (OR 5.6, 95% CI 3.4–9.0). In conclusion, noncompliance with hand hygiene was high in this hematology unit, especially among nurses and before activities. Variations with the type of HCW and activity suggest that targeted educational programs and feedback control may be useful.