The influence of the different extracts of propolis and chlorhexidine on mutans streptococci and the development of dental caries in rats was examined in this study. Specifically, the antimicrobial effects of propolis extracts (including ethanol, methanol, chloroform, hexane, propylene glycol, and ethyl acetate) on Streptococcus mutans UA159 and Streptococcus sobrinus 6715 were studied. Minimal inhibitory concentration and minimal bactericidal concentration were determined using the National Committee for Clinical Laboratory Standards broth macrodilution method. In the animal study, the rats were infected with S. sobrinus and fed a cariogenic diet. They were treated topically twice a day with both ethanol extract of propolis (EEP) and hexane extract of propolis (HEP) for 5 weeks. After the experimental period, both the microbial composition of their dental plaque and their caries score were assessed. EEP and HEP showed the highest antimicrobial activity against the tested microorganisms. The chemical composition of EEP and HEP were examined using gas chromatography-mass spectrometry. The pinobanksin and naringenin were the main phenolic compounds identified in both extracts. Further, both of the extracts significantly reduced the severity of smooth surface caries in vivo. The severity of the sulcal caries (Ds) was significantly reduced by EEP compared to the control group (P < 0.05). In conclusion, poplar propolis demonstrated antimicrobial activity against mutans streptococci, indicating that it may be used in caries prevention.