Purpose: This study compares in vitro remineralization potential and cytotoxicity of fluoride-free varnish combinations containing bioactive glass, eggshell, and membrane powder and fluoride varnish formulations on artificial caries lesions. Methods: Artificial caries lesions were formed in two windows on third molars. One of the windows was coated with one of the following varnish formulations: FV (fluoride varnish), F-BAGV (fluoride and bioactive glass containing varnish), BAGV (bioactive glass containing varnish), EPV (eggshell powder containing varnish), EMP–EPV (eggshell membrane protein and eggshell powder containing varnish), STMP-EMP–EPV (sodium trimetaphosphate-treated eggshell membrane protein and eggshell powder containing varnish). The samples were remineralized, then investigated under scanning electron microscopy, and elemental analyses were performed by X-ray dispersive analysis (EDX). In addition, the traditional colorimetric tetrazolium-based reduction assay (XTT) and the modern impedance-based real-time cell analysis system (RTCA) were used to investigate their cytotoxicity. Results: The varnish applied area’s Ca/P ratio was lower than stoichiometric hydroxyapatite except for EPV (1.66) and STMP-EMP–EPV (1.67) groups. Undiluted extracts of all varnishes, 1:2 dilutions of FV and F-BAGV groups were cytotoxic in XTT assay. In RTCA, the normalised cell index of the EMP–EPV and STMP-EMP–EPV groups was higher than the control group. Conclusion: Bioactive glass, eggshell, eggshell membrane proteins and STMP-treated eggshell and eggshell membrane protein containing varnish have similar remineralizing effect to fluoride-containing varnish on demineralized enamel. Integrating biological or bioactive components instead of fluoride into the dental varnishes might reduce cytotoxicity.