Aim: The study aimed to investigate the effect of a customary fluoride solution, containing sodium fluoride and amine fluoride, on initial biofilm formation on enamel and dentin in situ compared directly to chlorhexidine. Methods: Bovine enamel and dentin specimens were mounted on maxillary splints carried by 9 subjects. After 1 min of pellicle formation, rinses with tap water (control), chlorhexidine (meridol med CHX 0.2%, GABA) and a fluoride mouthrinse (elmex, GABA) were performed for 1 min. Subsequently, the slabs were carried for another 8 h. The adherent bacteria were determined by DAPI staining, live-dead staining and determination of colony-forming units after desorption; glucan formation was visualized with concanavalin A. Additionally, energy-dispersive X-ray spectroscopy (EDX) analysis of the in situ biofilm layers was conducted, and contact angle measurements were performed. Statistical evaluation was performed by means of the Kruskal-Wallis test followed by the Mann-Whitney U test (p < 0.05). Results: In the control group, significantly higher amounts of adherent bacteria were detected on dentin (4.8 × 10⁶ ± 5.4 × 10⁶ bacteria/cm²) than on enamel (1.2 × 10⁶ ± 1.5 × 10⁶ bacteria/cm², DAPI). Chlorhexidine significantly reduced the amount of adherent bacteria (dentin: 2.8 × 10⁵ ± 3.4 × 10⁵ bacteria/cm²; enamel: 4.2 × 10⁵ ± 8.7 × 10⁵ bacteria/cm²). Rinses with the fluoride solution also significantly reduced bacterial adherence to dentin (8.1 × 10⁵ ± 1.5 × 10⁶ bacteria/cm²). Fluoride could not be detected by EDX analysis of the biofilms. Fluoride mouthrinsing did not influence the wettability of the pellicle-covered enamel surface. Conclusion: In addition to the reduction of demineralization and antibacterial effects, fluorides inhibit initial biofilm formation on dental hard tissues considerably, especially on dentin.