Background: Matoa (Pometia pinnata) is a plant of the family Sapindaceae. Matoa fruit has antibacterial activity because it contains flavonoids, tannins and saponins, while the rind contains compounds matoa tannins, saponins and alkaloids. Utilization of matoa can be used as a hand sanitizer. This study aims to determine the influence of matoa (Pometia pinnata) hand sanitizer fruit peels to the number of gram-negative bacteria hand isolate. Metode: This study used a true experiment with a type of research study design pre-test and post-test. The subjects were janitor (Cleaning Staff) of University of Muhammadiyah Yogyakarta. The tools and materials were Mac Conkey media, incubator, hand sanitizer with matoa fruit peel extract 0.5% and 1%. The process by counting the number of gram negative bacteria before and after treatment. Data of germs number before-after treatment were analyzed using Wilcoxon, Kruskal-Wallis and Mann-Whitney test. Result: The lowest number of bacteria found on palms untreated at 750 CFU / cm2. Hand sanitizer of matoa fruit peel increased numbers of gram-negative bacteria, it was proved by the highest number of bacteria in giving the treatment of matoa fruit peel hand sanitizer 0.5% at 1541.75 CFU / cm2. Result of statistical analysis using Wilcoxon and Kruskal Wallis test at p>0,05, so this study proved that hand sanitizer matoa fruit peel with 0.5% and 1% concentration did not give influence in reducing the number of gram-negative bacteria hand isolates of cleaning staff. Conclusion: Hand sanitizer of matoa fruit peels did not give influence in reducing the number of Gram-negative bacterial hand isolate of cleaning staff. Matoa fruit peels hand sanitizers with a concentration of 0.5% and 1% did not give influence in reducing the number of gram-negative bacteria hand isolate of cleaning staff.