The treatments for dentin hypersensitivity (DH) may change the surface roughness of the root dentin, which can lead to biofilm accumulation, increasing the risk of root caries. The aim was to compare the surface roughness of root dentin after different treatments of DH and the biofilm formation on those surfaces. After initial surface roughness (Sa) assessment, 50 bovine root fragments received the following treatments (n=10): G 1-no treatment; G2-5% sodium fluoride varnish; G3-professional application of a desensitizing dentifrice; G4-toothbrushing with a desensitizing dentifrice; and G5-diode laser application (908nm; 1.5W, 20s). The Sa was reevaluated after treatments. Afterward, all samples were incubated in a suspension of Streptococcus mutans at 37°C for 24h. The colony-forming units (CFU) were counted using a stereoscope, and the results were expressed in CFU/mL. The one-way ANOVA and the Tukey's tests compared the roughness data and the results obtained on the bacterial adhesion test (α=5%). G2 (2.3±1.67%) showed similar Sa variation than G1 (0.25±0.41%) and G5 (5.69±0.99%), but different from group G3 (9.05±2.39%). Group 4 showed the highest Sa variation (30.02±3.83%; p<0.05). Bacterial adhesion was higher in G4 (2208±211.9), suggesting that bacterial growth is greater on rougher surfaces. The diode laser and the conventional treatments for DH may change the surface roughness of the root dentin, but only brushing with desensitizing dentifrice induced a higher bacteria accumulation on root dentin surface.