In this study, for the first time, we investigated in vitro inhibitory effects of Drymocallis rupestris extracts and their subfractions obtained with solvents of different polarity (aqueous, 50% ethanolic, diethyl ether, ethyl acetate and n-butanolic) against bacterial viability and caries virulence factors of Streptococcus spp. strains. The diethyl ether subfraction (PRU2) showed bacteriostatic and bactericidal activity against mutans streptococci, with minimum inhibitory concentrations (MICs) in the range of 0.75-1.5 mg/mL and minimum bactericidal concentrations (MBCs) in the range of 1.5-3 mg/mL. Furthermore, PRU2 inhibited biofilm formation by Streptococci in a dose-dependent manner. It was also found that all five D. rupestris preparations exhibited diverse inhibitory effects on de novo synthesis of water-insoluble and water-soluble α-d-glucans by glucosyltransferases of the mutans group streptococci. The phytochemical profile of investigated samples was determined by spectrophotometric and chromatographic (HPLC-DAD-MS3) methods. The high polyphenol (total phenol, phenolic acids, tannins, proantocyanidins, and flavonoids) contents were found which correlated with anticariogenic activity of the analyzed samples. The results demonstrate that D. rupestris extracts and their subfractions could become useful supplements for pharmaceutical products as a new anticariogenic agent in a wide range of oral care products. Further studies are necessary to clarify which phytoconstituents of D. rupestris are responsible for anticaries properties.