The objectives of the in vitro study were: (1) to investigate the effect of combining L-arginine (Arg) and NaF on the growth of Lactobacillus rhamnosus GG (LRG); and (2) to identify an optimum synergistic concentration for the synbiotic (Arg + LRG)-fluoride (SF) therapy. 1% Arg + 2000-ppm NaF (A-SF) and 2% Arg + 2000-ppm NaF (B-SF) demonstrated antagonism against LRG (FIC > 4.0). Both XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) assays showed that A-SF and B-SF enhanced the growth of LRG when compared to 2000-ppm NaF and LRG control. Colony forming units, bacterial weight, and biofilm thickness of A-SF and B-SF were significantly higher than 2000-ppm NaF and LRG control. Biofilm imaging depicted that 2000-ppm NaF inhibited biofilm formation; while 1%/2% Arg, A-SF, and B-SF increased biofilm growth of LRG. Lactic acid formation was the lowest for 2000-ppm NaF, followed by A-SF and then B-SF. The SF buffer potential after 24 h was the highest for B-SF, and then A-SF. Biofilm pH for B-SF was closest to neutral. Fluoride, Arg and LRG bioavailability remained unaffected in B-SF. The relative gene expression for arcA, argG, and argH was significantly higher for B-SF than the respective controls. In conclusion, combining 2% Arg, 2000-ppm NaF, and LRG provides an optimum synbiotic-fluoride synergism.