Background <p>This study compared in vitro cytotoxicity-related effects of four commercially available 5% sodium fluoride (NaF) varnishes (Enamelast^®, Profluorid^®, Polimo^®, Proshield^®) on human gingival fibroblast cells under standardized exposure conditions.</p> Methods <p>Varnish-derived supernatants were prepared using a standardized extraction approach and applied to HGF-1 cells at six concentrations ranging from 0.0625% to 2% for 24&#xa0;h. Cell viability, membrane integrity, and migration were assessed using MTT, LDH release, and wound-healing assays, respectively. Experiments were performed in three independent biological replicates with technical triplicates. Data were analyzed using two-way ANOVA followed by Tukey’s post hoc test for concentration-response assays and one-way ANOVA followed by Tukey’s test for migration analysis.</p> Results <p>All four varnishes showed concentration-dependent adverse effects on HGF-1 cells. Polimo^® showed the lowest IC₅₀ and the highest LDH release, indicating the least favorable in vitro profile among the tested materials. Enamelast^® showed the highest IC₅₀ and a comparatively milder response, while Proshield^® and Profluorid^® showed an intermediate profile. All varnishes significantly reduced cell migration compared with untreated control cells.</p> Conclusions <p>The four tested 5% NaF varnishes showed different cytotoxicity-related response profiles in HGF-1 cells, with Polimo^® showing the strongest adverse response and Enamelast.</p>