Background and Objectives: The etiology of nosocomial infections, the frequency of contaminated hands with the different nosocomial pathogens, and the role of health care workers' hands during outbreaks suggest that a hand hygiene preparation should at least have activity against bacteria, yeasts, and coated viruses. Hand washing is emphasized as the single most important measure to prevent cross transmission of micro-organisms and thus to prevent nosocomial infections. In this respect, we compared the antibacterial activity of handsept with decosept as two hygienic hand disinfections.Material and Methods: In vitro activity of “Handsept” (Isopropanol, N-propanol, Benzalkonium chloride) and “Decosept” (Propan-2-o, Benzyl- C12- 16- alkyl dimethyl ammonium, propan-1-01) were tested against 4 reference standard strain and 6 clinical isolated. Samples including Escherichia coli, Klebsiella spp, Staphylococcus aureus, Pseudomonas spp. In vitro activity was established using broth dilution method, additionally; the in vivo anti-microbial properties of the two antiseptic agents were tested on the bacterial contaminated hands of volunteers.Results: Our results showed that “Handsept” can prevent the growth of 10 bacterial strains after 15, 30, 40 seconds. In another study “Handsept” was applied to contaminated hands of the health care workers with (106 /ml) of bacterial suspensions, which after 15 seconds no bacterial was found on their hands.Conclusion: The use of hygienic hand disinfection (e.g. Handsep and Decosept) in all these situations will be effective in preventing cross-transmission of nosocomial pathogens. Similar findings have been found for “Handsept” and “Decosept”. Thus we suggest using “Handsept” instead of “Decosept” in hospitals given their efficacy is similar and “Hansept” produced in Iran and has a more reasonable pricing than “Decosept”.