Oral biofilms, which are known as dental plaque, are the reason for a wide range of oral and systemic diseases, which contribute to serious health risks. Paeoniae Radix Alba (PRA) is traditionally used as a folk medicine with anti-inflammatory, cardioprotective, and hepatoprotective properties. PRA is currently used in a variety of therapeutic approaches for oral diseases. Nevertheless, its inhibitory effect on oral biofilm formation and the basis for its efficacy have not been clarified. This study intended to screen the potential compounds in PRA that inhibit oral biofilm formation using biochromatography. Two biofilm models based on S. mutans were used to determine the inhibitory effect of PRA on biofilm formation. The extraction of PRA was divided into fractions with different polarity, the active fraction screened, and an HPLC profile constructed for the active fraction. Three potential compounds were screened using targeted oral biofilm extraction, and subsequent validation of the efficacy indicated that albiflorin is the main compound in PRA exerting anti-biofilm activity. Our results have revealed the pharmacological substance basis of PRA in inhibiting the formation of oral biofilm and provide a reference for the further use of PRA in the development of oral health products.