The presentin situstudy investigated the influence of a preparation containing CPP/ACP (caseinphosphopeptide-amorphous calcium phosphate) (GC Tooth mousse) on initial bacterial colonization of enamel and dentin. Therefore, pellicle formation was performedin situon bovine enamel and dentin specimens fixed to individual upper jaw splints worn by 8 subjects. After 1 min of pellicle formation GC Tooth mousse was used according to manufacturer’s recommendations. Rinses with chlorhexidine served as positive controls. Specimens carried without any rinse served as negative controls. After 8 h overnight exposure of the splints, bacterial colonization was quantified by fluorescence microscopy (DAPI and BacLight live/dead staining). Additionally, the colony forming units (CFU) were determined after desorption. Furthermore, the effects onStreptococcus mutansbacteria were testedin vitro(BacLight). There was no significant impact of CPP/ACP on initial bacterial colonization proved with DAPI and BacLight. Determination of CFU showed statistical significance for CPP/ACP to reduce bacterial adherence on enamel. Thein vitroinvestigation indicated no antimicrobial effects for CPP/ACP onStreptococcus mutanssuspension. Under the chosen conditions, CPP/ACP (GC Tooth mousse) had no significant impact on initial biofilm formation on dental hard tissues. The tested preparation cannot be recommended for biofilm management.