Aim: Gold nanorods (GNRs) have gained interest as a promising carrier for antibiotics. Gold nanorods may reduce the development of antimicrobial resistance in certain microbial species. Although applications of GNRs to mitigate oral biofilms are under development, their use in the oral cavity may have adverse effects. The aim of this study was to evaluate the potential penetration of GNRs into the tooth enamel structure using confocal laser scanning microscopy (CLSM) and scanning transmission electron microscopy (STEM). Materials and methods: Our approach was to synthesize GNRs with cationic [cetyltrimethylammoniumbromide (CTAB)] and anionic [11-mercaptoundecanoic acid (MUDA)] surface coatings. We hypothesized that penetration would be surface coating-dependent. Results: Regardless of the chemical modification of the GNRs of size ∼20 nm × 8 nm, exposure of these materials did not result in superficial penetration into the enamel. Conclusion: Within the limitations of this study, it is concluded that the use of CLSM and STEM is a feasible approach to investigate the penetration of nanomaterials into the tooth structure. Clinical significance: Exposure of the enamel with chemically modified GNRs of size ∼20 nm × 8 nm will not result in superficial penetration into the enamel.