In this study, were evaluated two extraction methods for obtaining theantimicrobial active fraction from ethanol extract of leaves of Isertia laevis: thefirst method of fractionation was the technique of Vacuum ColumnChromatography (VCC), which were used as FE: RP-18 and FM: MeOH: H2O,MeOH, MeOH:CH2Cl2 and CH2Cl2. The second method was ContinuousFractionation Liquid/liquid with solvents in order of increasing polarity (petrol,CH2Cl2, AcOEt, BuOH). In the two methods were the fractions with inhibitoryactivity against S. mutans ATCC 25175 and S. sobrinus. In the VCC, the activefractions were fraction MeOH, fraction MeOH-CH2Cl2 and the fractionationliquid/liquid was fraction CH2Cl2. Bioautography was used as an alternativemethod in the evaluation of the fractions, resulting in the inhibition of fractionMeOH and fraction CH2Cl2. Through Qualitative chemical was found that theactive fractions containing terpenoids, steroids and saponins. The two methodsallowed the separation of the antimicrobial active fraction of the plant, showingsignificant differences in cost, quantity and degree of mixing obtained alsodemonstrated the reproducibility and found in the separation of the activefraction and its action.