Streptococcus mutans, a common oral bacterium, often persists as a biofilm on teeth and contributes to the development of dental caries. In this study, we investigated the efficacy of ClyR, a chimeolysin (i.e engineered bacteriophage lysin) with an extended streptococcal host-range, against S. mutans biofilms in physiological and cariogenic conditions. Susceptibility tests demonstrated that ClyR was active against all clinical S. mutans isolates tested as well as S. mutans biofilms that were resistant to penicillin. S. mutans biofilms formed on hydroxyapatite discs, the mineral component of tooth enamel, in physiological or cariogenic sugar conditions were reduced by ~3 logs and 2 logs after treatment with 200 μg/ml ClyR, respectively. In comparison, only 1 log reduction was observed in the chlorhexidine (ChX) treated group and no killing effect was observed in the NaF treated group. A mouse model showed that repeated use of ClyR for 3 weeks (5 μg/day) reduced the number of colonized S. mutans in the dental plaques significantly (p<0.05), without harmful effects on the body weight and the vitality of mice. Furthermore, ClyR-specific antibodies could be detected in mouse sera but not saliva, suggesting there was no antibody neutralization after repeated use of ClyR in the oral cavity. Our data collectively demonstrates that ClyR is active against S. mutans biofilms in vitro and in vivo, thus representing a preventative agent for dental caries.