Objective: To investigate the effects of laser-treated enamel surface roughness on the adhesion of Streptococcus mutans. Methods: A total of 176 premolars extracted for subtractive orthodontic treatment were collected. Samples were randomly divided into four groups: control group, 35% phosphoric acid, Er:YAG laser, Er,Cr:YSGG laser. Surface roughness was detected using three-dimensional white light interferometric surface topography (n = 8/group), and surface morphology was observed by scanning electron microscope [(SEM), n = 8/group]. Samples were cultured in S. mutans solution for 12 h, 1 day, and 2 days. The colony forming units on the enamel surface were calculated (n = 8 per time point). The biofilm and activity of S. mutans were observed by SEM (n = 2/group) and confocal laser scanning microscope (n = 2/group). Correlation analysis was carried out between bacterial adhesion on the enamel surface at different time points and the surface roughness of the enamel treated by different methods. Results: After Er:YAG and Er,Cr:YSGG laser etching, the surface roughness of enamel increased (p < 0.05), but there was no difference in S. mutans adhesion and biofilm morphology after 1 day compared with traditional phosphoric acid etching (p > 0.05). S. mutans adhesion was positively correlated with the enamel roughness in the early stage. Conclusions: Er:YAG and Er,Cr:YSGG laser etching may be an alternative to traditional phosphoric acid etching.