It is well established that fluoride increases enamel resistance to acidic pH and promotes remineralization but its effect on bacterial composition and metabolism is not fully understood. In the current manuscript, we have grown in vitro oral biofilms and used 16S rRNA gene Illumina sequencing to study the effect of fluoride on DNA- and RNA-based bacterial composition. In addition, a metatranscriptomic approach has also been performed, in which the total RNA was copied to cDNA and sequenced in order to study gene expression profiles in the presence/absence of 500 ppm fluoride. Our data show a clear shift in total (DNA-based) and metabolically active (RNA-based) bacterial composition when biofilms are exposed to fluoride. Streptococcus oralis was the species most affected by fluoride exposure, with a 10-fold reduction in both DNA and RNA samples, whereas Rothia mucilaginosa underwent a 8-fold increase in the DNA and S. salivarius a 4- and 5-fold increase in the RNA and DNA samples, respectively. The metatranscriptome indicated that fluoride exposure induced a dramatic shutdown of sugar metabolism, including significant under-expression of different sugar transporters, fucosidases and a pyruvate oxidase, among others. Another gene with a significant under-expression in the presence of fluoride was the arginine deiminase, involved in ammonia production from arginine. In conclusion, the reduction in saccharolytic organisms and the inhibition of sugar fermentation pathways by fluoride may therefore be considered instrumental for the beneficial effect of fluoride-containing oral hygiene products.