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This study’s objective was to examine L-arginine (L-arg) supplementation’s effect on mono-species biofilm (<i>Streptococcus mutans</i>/<i>Streptococcus sanguinis</i>) growth and underlying enamel substrates. The experimental groups were 1%, 2%, and 4% arg, and 0.9% NaCl was used as the vehicle control. Sterilised enamel blocks were subjected to 7-day treatment with test solutions and <i>S. mutans</i>/<i>S. sanguinis</i> inoculum in BHI. Post-treatment, the treated biofilms stained for live/dead bacterial cells were analysed using confocal microscopy. The enamel specimens were analysed using X-ray diffraction crystallography (XRD), Raman spectroscopy (RS), and transmission electron microscopy (TEM). The molecular interactions between arg and MMP-2/MMP-9 were determined by computational molecular docking and MMP assays. With increasing arg concentrations, bacterial survival significantly decreased (<i>p</i> < 0.05). The XRD peak intensity with 1%/2% arg was significantly higher than with 4% arg and the control (<i>p</i> < 0.05). The bands associated with the mineral phase by RS were significantly accentuated in the 1%/2% arg specimens compared to in other groups (<i>p</i> < 0.05)<b>.</b> The TEM analysis revealed that 4% arg exhibited an ill-defined shape of enamel crystals. Docking of arg molecules to MMPs appears feasible, with arg inhibiting MMP-2/MMP-9 (<i>p</i> < 0.05). L-arginine supplementation has an antimicrobial effect on mono-species biofilm. L-arginine treatment at lower (1%/2%) concentrations exhibits enamel hydroxyapatite stability, while the molecule has the potential to inhibit MMP-2/MMP-9.