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The hands of healthcare workers (HCWs) are the most common vehicle for the transmission of micro-organisms from patient to patient and within the healthcare environment. The aim of this study was to evaluate the impact of a multimodal campaign on the type and amount of resident and transient flora and the presence of potential risk factors for hand contamination during routine care. A before-after (PRE and POST periods) interventional study was carried out in medical wards of a tertiary care hospital. Eighty-nine samples were analysed. Samples were cultured immediately before patient contact using a glove-juice method. Data collected included socio-demographic and risk factors for hand contamination. Flora was measured as log10 c.f.u. ml(-1) and evaluated by comparing median values in the PRE and POST periods. Transient flora was isolated from the hands of 67.4 and 46.1 % of HCWs in the PRE and POST periods, respectively (P<0.001). Enterobacteriaceae, Pseudomonas spp. and meticillin-sensitive Staphylococcus aureus were the predominant contaminants. Resident flora was isolated from 92.1 % of HCWs in the PRE period and from 70.8 % in the POST period (P<0.001). The meticillin-resistant coagulase-negative staphylococci log10 c.f.u. count ml(-1) decreased from 1.96 ± 1.2 to 0.89 ± 1.2 (mean ± s d; P<0.001), and the global flora count decreased from 2.77 ± 1.1 to 1.56 ± 1.4 (P<0.001). In the POST period, the wearing of fewer rings (P<0.001), shorter fingernail length (P = 0.008), a shorter time since recent hand hygiene (HH) (P = 0.007) and an increased use of alcohol-based hand rub instead of soap (P<0.001) were documented. The HH multimodal strategy reduced the number of risk factors and the level of HCW hand contamination.