The bovine milk protein osteopontin (OPN) has been shown to reduce the adhesion of oral bacteria to saliva-coated surfaces, which reduces biofilm formation and may contribute to caries control. We now quantified the effect of OPN (Lacprodan OPN-10) treatment on the adhesion force of Lactobacillus paracasei subsp. paracasei and Actinomyces naeslundii using atomic force microscopy based single cell force spectroscopy (AFM-SCFS). Furthermore, we performed a fluorescent lectin binding analysis (FLBA) to investigate if OPN treatment resulted in the selective blocking of specific cell surfaces glycoconjugates of L. paracasei, A. naeslundii and Streptococcus mitis. For AFM-SCFS, saliva-coated tipless AFM cantilevers were approached on single cells. The cantilevers were then retracted and adhesion was quantified from the recorded force curves. Adhesion on the same cells (n=10) was compared before and after OPN treatment. Adhesion energy was found to be reduced by 94% for L. paracasei and 61% for A. naeslundii (p<0.05). For FLBA, the binding of 75 FITC-labelled lectins to the three bacteria was screened. Lectins BanLec, ConA, VGA and WGA bound well to A. naeslundii, lectins ABA and HPA to L. paracasei, and lectins VGA and WGA to S. mitis. Immobilized bacteria were incubated with these lectins in the presence and absence of OPN. For each combination, 12 confocal images were acquired with fixed microscope settings, and average fluorescence intensities were determined. Experiments were performed in triplicate and analyzed with two-sample t-tests. No statistically significant differences were observed between OPN-treated and OPN-free cells (p>0.05). OPN strongly reduces the adhesion force of oral bacteria to saliva-coated surfaces, but the effect cannot be ascribed to the selective blocking of cell surface glycoconjugates.