Using miswak is one of sunnah performed by Prophet Muhammad SAW. The wood of the miswak is used to clean the teeth because it contains a variety of compounds including these terpenoids, alkaloids, tannins and flavonoids which function as antibacterial on the periodontal disease because of the bacterial infection. One of them is the bacteria Staphylococcus aureus. Therefore it needs to be done the extraction and fractionation process to get the active compounds in wood miswak that has antibacterial activity against Staphylococcus aureus. This research is experimental research with Complete Random Design methods. Ethanol extract separated using miswak from solvent of ether, chloroform and ethanol. The methods used for screening phytochemicals in thin Layer Chromatography (TLC) and test tube using the reagents and the test of antibacterial activity is done using a concentration of 50% v/v, 25% v/v, 12.5% v/v and 6.25% v/v with the hole diffusion method, metronidazole as a positive control and a negative control as DMSO. The data obtained are then analyzed using One Way ANOVA test. Based on the results of the ANOVA analysis it can be concluded that secondary metabolites contained in miswak wood fraction of ether, chloroform and ethanol is terpenoid, alkaloid, tannin and flavonoid. Ethanol extract of miswak fraction from ether, chloroform and ethanol have antibacterial activity against Staphylococcus aureus. The zone of inhibition of ethanol fraction contained on the smallest 6.25% v/v with the inhibitory zones amounted to 11.6 mm and the greatest inhibition zones are present in the ether fraction of 50% v/v with the drag of 20.83 mm zone. aureus Keywords: Antibacterial, Fractionation, Miswak, Staphylococcus