Background: The present study aimed to investigate if mouthrinses with different types of bovine milk or milk protein isolates influence the initial bacterial colonization of the tooth surface. Methods: From 8 subjects, different biofilm samples were collected in situ on bovine enamel slabs: after 3 min of pellicle formation, mouthrinses with homogenized UHT-milk (0.3% and 3.5% fat), homogenized fresh milk (3.5% fat), non-homogenized milk 3.8%, 30% UHT-cream or a 3% micellar casein isolates containing preparation were performed, followed by a continued intraoral slab exposure for 8 h overnight. As control, no rinse was adopted. Afterwards, bacterial adhesion was quantified by DAPI staining and bacterial viability was determined by BacLight LIVE/DEAD-staining. Extracellular polysaccharides were visualized by Concanavalin A/Alexa-Fluor 594-staining. Statistical analysis was performed by the Kruskal-Wallis test and the Mann-Whitney U test followed by Bonferroni-Holm correction. Results: After 8 h of intraoral biofilm formation, 1.62*10⁶±1.68*10⁶ bacteria/cm² were quantified in the control samples. Viability staining showed a distribution of 35% vital to 65% avital bacteria. None of the applied mouthrinses showed a significant change (p > 0.01) in bacterial colonization. A tendency to reduce bacterial colonization in situ was observed for non-homogenized milk and casein micelles. Conclusion: Mouthrinsing with bovine milk and milk protein isolates had no significant impact on initial biofilm formation at the tooth surface. Clearly, it does not increase bacterial colonization.