To examine the effect of incorporating arginine in a 5% sodium fluoride (NaF) varnish on its remineralization potential and HGF-1 cytotoxicity. Experimental varnishes were prepared using two arginine variants: l-arginine (Arg) and l-arginine monohydrochloride (Arg.HCl) at 2%, 4%, & 8% w/v. with control 5% NaF varnish. Sound enamel specimens with varnish treatments were subjected to a 4-day demineralization assay and surface roughness analysis using AFM (n = 3). Enamel specimens with artificial incipient caries-like lesions were subjected to a remineralization assay (n = 6) and an 8-day pH-cycling (n = 3). For remineralization assay, enamel F/Arg-uptake and Ca/PO43-i-content were estimated. During pH-cycling, EDX mapping for Ca/F-content and mineral density using micro-CT were assessed. The HGF-1 cytotoxicity of the varnishes was examined using MTT/CCK-8 assay. The Ca-content/F-uptake of the 2% Arg-NaF varnish treated specimens were significantly higher than other varnishes (p < 0.05). The 2% Arg-NaF had significantly higher remineralization potential than NaF (p < 0.001), suggesting Arg-Ca-F complex formation post-varnish application. The 8% Arg-NaF exhibited significantly more pronounced cytotoxicity to HGF-1 cells than 2% Arg-NaF (p < 0.001) and NaF varnish. Incorporation of 2% Arg in NaF varnish enhanced the enamel remineralization potential of NaF varnish; while 8% Arg in NaF varnish was cytotoxic to HGF-1. A professionally deliverable l-arginine-enriched NaF varnish for caries prevention addresses a global public health priority. The synergism between l-arginine and NaF varnish counters the limited antimicrobial effects of fluorides and supports acquisition of a balanced oral microbiome. The combined arginine-fluoride varnish will provide the much-needed ecological-based approach for caries prevention.