A strain of lactic acid bacteria from cheese was isolated, that showed strong growth inhibitory effects onStreptococcus mutans. The API 50CH system and 16S rDNA sequencing verified that this was a novel strain, and was namedLacticaseibacillus rhamnosusVHProbi M14. The strain inhibited the growth ofS.mutansandFusobacterium nucleatumunder mixed culture conditions, coaggregated withS.mutansandF.nucleatum, and reduced the adhesion ofS.mutansandF.nucleatumon cultured human primary gingival epithelial (HPGE) cells. The pH, peroxidase and protease sensitivity testing found antibacterial substances of protein- and peptide-like structures in addition to organic acids. The antimicrobial substances were sensitive to hydrolysis with trypsin, papain and pineapple protease and were inactived at temperatures above 100°C. Ammonium sulphate-precipitated proteins from the M14 strain retained the ability to inhibit the growth ofS.mutansandF.nucleatum. The M14 strain contained 23 bacteriocin-related genes encoding for metabolites, belonging to class II bacteriocins. The M14 strain also showed inhibitory effects on 8 other pathogenic strains (A.actinomycetemcomitans,C.albicans,E.coli,G.vaginalis,P.acnes,P.gingivalis,S.aureus,S.enteritids), and thus has a broad spectrum of bacterial inhibition. This new isolate has been identified as having potential to be used as a probiotic bacterium in clinical applications.