The observance of hand hygiene compliance is important to reduce cross-infection by micro-organisms. The aim of this preliminary study was to evaluate the level of hand hygiene in healthcare workers from different departments, with particular emphasis on transient flora. The study was conducted in three departments (Surgery, Intensive Care Unit, Obstetrics and Gynecology) of a hospital in Campania, southern Italy. Over a six-month period, 50 healthcare workers were randomly tested. Imprints of palms and fingertips were taken monthly during the morning shift. The number of colonies per plate was counted and transient pathogens were identified. Risk factors for hand contamination were determined. Total flora was found in the following CFU means per palm and per five fingertips (95% CI): Obstetrics and Gynecology [palms 130 CFUs (95% CI 85-180); fingertips 125 CFUs (95% CI 92-160)]; ICU [palms 80 CFUs (95% CI 58-99); fingertips 62 CFUs (95% CI 45-82)]; Surgery [palms 75 CFUs (95% CI 41-120); fingertips 70 CFUs (95% CI 52-90)] Transient flora was found on 39% of healthcare workers' hands. The only factor associated with hand contamination by transient flora was the absence of gloving during healthcare procedure (P = 0.02).