The microorganisms in the hand cause the spreading of nosocomial infection. Washing hands is the most effective way to control infection and it is important to know the effectiveness of antiseptic hand sanitizer to reduce bacteria growth in the hands. This study determine effectivity of 70% alcohol, 4%Clorhexidine Gluconate soap and Irgasan DP 300 and to know most powerful in reducing number of bacteria. Research methode quasi – experimental on one group (one group pre test – post test design). There are 3 groups consist of 20 healthcare worker each group. The sample is taken from hand before and after hand washing with hand sanitizer, then be cultured in Trypticase Soy Agar (TSA) media with pour plate methode during 24 hours in 37oC, and count the number of bacteria. Data analyzed by paired sample T test and One way ANOVA. The average number of bacteria before hand washing with 4% Clorhexidine Gluconate Soap, 70% Alcohol and Irgasan DP 300 are 161.9 cfu/cm2 , 168.7 cfu/cm2 , and 129.6 cfu/cm2 respectively, and after hand washing are 47.4 cfu/cm2 , 45.6 cfu/ cm2, 69.7 cfu/cm2 respectively. The average of reducing bacteria growth after hand washing by 4% Clorhexidine Gluconate Soap is 69.4%, by 70% Alcohol is 72.61% and by Irgasan DP 300 is 46.80 %. The p-value in statistic T paired sample test is 0.00 and ANOVA test is 0.01. There are differences between 3 hand sanitizer to reduce number of bacteria in hands. 70% Alcohol is most effective to reduce number of bacteria and to prevent spreading of nosocomial infection.