In the present study, an original chlorhexidine-loaded nanocapsule-based gel (Nanochlorex) was tested as hand rub gel against the resident skin flora in comparison with 2-propanol 60% (v/v) and 62% (v/v) ethanol-based gel (Purell). After 30-s hand rub, the immediate bactericidal effect of Nanochlorex was found comparable to 2-propanol 60% (v/v) (reduction factor, RF: 0.30+/-0.35 versus 0.38+/-0.55, P>0.05) against aerobic bacteria, whereas the post-values of surviving anaerobes were shown significantly lower from Nanochlorex (P<0.001) and insignificant from 2-propanol 60% (v/v) (P>0.05). Sustained antibacterial effect of Nanochlorex was confirmed against the resident and transient hand flora in two sets of experiment. In the first, the results obtained with the glove-juice technique showed that the bactericidal effect induced by Nanochlorex hand rub persisted throughout 3-h period, while Purell failed to reduce significantly the post-values of surviving bacteria. In the second, repeated artificial contaminations with Staphylococcus epidermidis was carried out onto ex vivo human skin pre-treated by either Nanochlorex or Purell for 5min, then maintained in cell diffusion apparatus for 4h. The log(10) reduction of surviving bacteria was significantly higher with Nanochlorex than that determined with Purell after three successive contaminations (from approximately 5.5 to 1.5 log(10) reduction for Nanochlorex between the first and the third contamination; approximately 1log(10) reduction for Purell throughout the experiment), confirming the sustained antibacterial effect of chlorhexidine-loaded nanocapsule-based gel. The immediate and sustained antibacterial effect of Nanochlorex was explained by chlorhexidine carrier system which improved the drug targeting to bacteria and reduced from osmotic gel further bacterial growth on the skin. Nanochlorex) might constitute a promising approach for hygienic hand disinfection in care practice performing multiple procedures.