Zirconia is becoming a prevalent material in dentistry. However, any foreign bodies inserted may provide new niches for the bacteria in oral cavity. The object of this study was to explore the effect of surface properties including surface roughness and hydrophobicity on the adhesion and biofilm formation of Streptococcus mutans (S. mutans) to zirconia. Atomic force microscopy was employed to determine the zirconia surface morphology and the adhesion forces between the S. mutans and zirconia. The results showed that the surface roughness was nanoscale and significantly different among tested groups (P < 0.05): Coarse (23.94 ± 2.52 nm) > Medium (17.00 ± 3.81 nm) > Fine (11.89 ± 1.68 nm). The contact angles of the Coarse group were the highest, followed by the Medium and the Fine groups. Increasing the surface roughness and hydrophobicity resulted in an increase of adhesion forces and early attachment (2 h and 4 h) of S. mutans on the zirconia but no influence on the further development of biofilm (6 h~24 h). Our findings suggest that the surface roughness in nanoscale and hydrophobicity of zirconia had influence on the S. mutans initial adhesion force and early attachment instead of whole stages of biofilm formation.