OBJECTIVE To compare the in vitro cytotoxicity of four commercial topical fluoride varnishes widely used in daily dental practice for the prevention of caries on human fibroblasts: Cervitec F, Fixofluor, Fluor Protector S and Duraphat. METHODS Human gingival fibroblasts (hGF) were exposed to different concentrations of fluoride varnishes extracts. Biological assays, including MTT and IC50 value determination, annexin-V/7-AAD staining, cell migration and F-actin staining with phalloidin were carried out. Statistical analyses were performed using one-way ANOVA and Tukey's post hoc test. RESULTS At 4% concentration, all of the fluoride varnishes extracts affected fibroblasts metabolic activity, exhibiting a high degree of cytotoxicity at all measured time points. At 0.1% and 1%, Duraphat and Fixofluor or Fluor Protector S and Cervitec F exerted the lowest or highest cytotoxic effects, respectively. Similar effects were evidenced when induction of apoptosis/necrosis and cell migration assays were analyzed. Immunocytochemical assays revealed a similar number of fibroblasts, without changes in the morphology and F-actin content at 0.1% concentration of all tested materials, while at 1% concentration, Fluor Protector S and Cervitec F showed few cells with aberrant morphology or non-adhered cells, respectively. CONCLUSIONS Different commercial topical fluoride varnishes with the same therapeutic indication may exhibit different biological effects and cytotoxicity on fibroblasts.