Dental surface conditioning by Er:YAG laser is currently being investigated, as not all of the mechanisms and effects of this technique have been clearly studied. Thus, the aim of the present study was to assess the cervical microleakage of Class II resin composite restorations in endodontically treated teeth following either the respective conventional conditioning or additional Er:YAG laser conditioning, in association with varied adhesives. Standardized mesial-occlusal-distal cavities (two gingival walls positioned in dentin and enamel, respectively) were created in 60 extracted human premolar teeth. Following the completion of the endodontic therapy, the teeth were grouped into six categories based on conditioning modality and adhesive strategy as follows: group 1-37% phosphoric acid/Adper Single Bond 2 (ASB2); group 2-Er:YAG laser/37% phosphoric acid/ASB2; group 3-Clearfil SE Bond (CSE); group 4-Er:YAG laser/CSE; group 5-Adper Easy One (AEO); and group 6-Er:YAG laser/AEO. Specimens were submitted to thermocycling and dye penetration, followed by longitudinal sectioning. The dye penetration was evaluated using a stereomicroscope. One specimen from each group was assessed under a scanning electron microscope for adhesive interface analysis. No significant differences were found between the conditioning modalities, nor between the adhesive systems at both margins. Groups 1 and 2 showed a lower degree of microleakage in the enamel vs. dentin (p = 0.002). Group 2 showed a significantly lower incidence of microleakage in enamel vs. dentin (p = 0.005). CSE and AEO were comparable with that of ASB2 regarding sealing ability. Additional Er:YAG laser conditioning may be beneficial before ASB2 application in enamel.