Recently, two chemically different types of Brazilian propolis (type-3 and -12) were shown to have cariostatic properties. This study aimed to evaluate the influence of their isolated fractions on mutans streptococci viability, glucosyltransferases (GTFs) activity and caries development in rats. The ethanolic extracts of propolis (EEPs) were serially fractionated into hexane (H-fr), chloroform, ethyl acetate, and ethanol. The ability of the four fractions and EEP to inhibit Streptococcus mutans and Streptococcus sobrinus growth and adherence to a glass surface was examined. The effect on GTFs B and C activity was also determined. For the caries study, 60 Wistar rats infected with Streptococcus sobrinus were treated topically twice daily as follows: (1) EEP type-3, (2) H-fr type-3, (3) EEP type-12, (4) H-fr type-12, and (5) control. In general, the H-fr from both types of propolis showed the highest antibacterial activity and GTFs inhibition. Furthermore, the EEP and H-fr type-3 and -12 were equally effective in reducing dental caries in rats. The data suggest that the putative cariostatic compounds of propolis type-3 and -12 are mostly non-polar; and H-fr should be the fraction of choice for identifying further potentially novel anti-caries agents.