The purpose of this study was to evaluate persistent activity of three alcohol-based antiseptics widely used in the clinical routine containing chlorhexidine, triclosan or mecetronium after hand disinfection. Four tests were used to evaluate the antimicrobial activity of antiseptics on: (i) resident microbiota; (ii) nutrient agar plates (NAP) subsequently inoculated with a test organism; and transient microbiota acquired by contact with dry hands (iii), or (iv) a wet inert surface that had been artificially contaminated. Four reference strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis) and an Acinetobacter baumannii strain responsible for a hospital outbreak were used as transient microbiota. Antimicrobial activity was calculated according to the CFUs reduction by reference to non-disinfected control hands. The antiseptics were applied according to European Standard EN1500. Solutions containing chlorhexidine or triclosan showed some persistent effects on transient microbiota on inert humid surfaces and NAP, but not on contaminated dry hands. Solutions containing mecetronium showed no persistent effect on transient flora in any of the trials. All alcohol-based antiseptics tested were more effective against resident flora than soap. No persistent activity was observed for A. baumannii in any of the trials. Chlorhexidine and triclosan are preferred when an antiseptic with persistent activity is desired, but a moist environment appears to be necessary for that antibacterial activity. Accordingly, relevant conclusions regarding the persistent activity of antiseptics for clinical practice and protection against bacterial contamination cannot be derived from this study for the alcoholic solutions tested.