The microorganisms in the hand can cause the spreading of nosocomial infection. Washing hands is the most effective way to control infection and it is important to check the effectiveness of antiseptic hand sanitizer to reduce bacteria growth in the hands. This study determine effectivity of 70% alcohol, Clorhexidine Gluconate 4% soap and Irgasan DP 300 and to know most powerful of theme in reducing bacteria growth. 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 TSA media 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 Clorhexidine Gluconate 4% Soap, 70% Alcohol and Irgasan DP 300 are 161.9 cfu/ml, 168.7 cfu/ml, and 129.6 cfu/ml respectively, and after hand washing are 47.4 cfu/ml, 45.6 cfu/ml, 69.7 cfu/ml respectively. The average of reducing bacteria growth after hand washing by Clorhexidine Gluconate 4% 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 bacteria growth in hands . Alcohol is the best hand sanitizer to reduce bacteria growth and to prevent spreading of nosocomial infection