OBJECTIVES: The study aimed to develop and evaluate in vitro a varnish containing fluoride-doped glass ceramics capable of inhibiting oral microorganisms, releasing hydroxyapatite-forming ions, and ensuring biocompatibility. MATERIALS AND METHODS: For the production of the experimental varnish (VE), composed of hydrogenated rosin, 10% and 20% by weight of glass ceramics with composition S53P4 (CF0) and doped with F- ions (CF5 and CF10) were incorporated. The EVs were characterized by Rheology, Scanning Electron Microscopy (SEM), FTIR, ion release (fluoride and calcium ions), cytotoxicity on VERO cells, and antimicrobial effect on S. mutans, S. aureus and C. albicans and anti-biofilm effect on S. mutans. The data were analyzed by One-Way ANOVA and Kruskal-Wallis test (P = 0.05). RESULTS: Homogeneous varnishes with good viscosity were obtained. Varnishes with 20% CF demonstrated biocompatibility and minimum inhibitory concentration (MIC) at concentrations lower than 10 mg/mL and 3.12 mg/mL, respectively, except for C. albicans. An anti-biofilm effect on S. mutans was observed for the varnishes with 20% CF. All varnishes with CF released more F- than the commercial varnish, with V20CF10 standing out, which released 4 times more F- ions in a quarter of the time. CONCLUSIONS: The V20CF10 varnish is a promising material for dental use in the treatment of early caries lesions, due to its biocompatibility, antimicrobial properties, and release of hydroxyapatite-forming ions. CLINICAL RELEVANCE: The use of a new varnish that combines antimicrobial properties with hydroxyapatite-forming ion release may prevent or halt the progression of dental caries lesions, offering greater efficacy than currently available varnishes.