Dental caries is a common disease affecting quality of life globally. In the present study, we found that a bacteriophage lysin LysP53 against Acinetobacter baumannii possesses selective activity on Streptococcus mutans, the main etiological agent of dental caries, even in low pH caries microenvironments, whereas only minor LysP53 activity was detected against Streptococcus sanguinis, Streptococcus oralis, and Streptococcus mitis. Testing activity against S. mutans planktonic cells showed that 4 μM LysP53 could kill more than 84% of S. mutans within 1 min in buffer with optimal pHs ranging from 4.0 to 6.5. Daily application of LysP53 on biofilms formed in BHI medium supplemented or not with sucrose could reduce exopolysaccharides, expression of genes related to acid resistance and adhesion, and the number of live bacteria in the biofilms. LysP53 treatment also showed similar effects as 0.12% chlorhexidine in preventing enamel demineralization due to S. mutans biofilms, as well as effective removal of S. mutans colonization of tooth surfaces in mice without observed toxic effects. Because of its selective activity against main cariogenic bacteria and good activity in low pH caries microenvironments, it is advantageous to use LysP53 as an active agent for preventing caries.