Microinvasive dentistry is based on the treatment of early carious lesions with the use of dental infiltrants. The commercially available Icon dental infiltrant does not contain any bacteriostatic component. An experimental preparation enriched with the missing component was synthesised. The aim of this study was to evaluate the cytotoxicity of the experimental preparation. Mouse fibroblasts of the L-929 lineage were used for the in vitro study. Cell morphology and viability were assessed. In the cytotoxicity analysis, it was shown that the experimental preparation (42.8 ± 10.3) after 24 h at two-fold dilution showed similar cytotoxicity to Icon (42.7 ± 8.8) (p > 0.05), while at four-fold dilution experimental preparation (46.7 ± 3.1), it was less toxic than Icon (34.2 ± 3.1) (p < 0.05). The experimental preparation has the potential to provide an alternative to the Icon commercial preparation. Further research is needed to evaluate the cytotoxicity of the experimental preparation over a longer period of time.