Purpose This in vitro study aimed at evaluating the influence of Nd:YAG irradiation and chlorhexidine (CHX) application on immediate and long-term bond strength of a self-etching adhesive system to dentin. Methods Thirty fragments of dentin were randomly divided into three groups according to the treatment to be applied to the dentin surface prior to applying the self-etching adhesive system (n = 10): CHX:2% CHX application (60 s); Nd: YAG (100 mJ/20 Hz for 20 s); and NT: no treatment. The adhesive system (Clearfil SE Bond, Kuraray) was applied according to the manufacturer's instructions, and composite resin restorations were made on the dentin. After 24 h, at 37°C, the resin-tooth blocks were sectioned perpendicular to the adhesive interface in the form of sticks (0.8 mm 2 of adhesive area), and randomly subdivided into two groups according to the microtensile bond strength (μTBS) test periods: immediately or 6 months after storage in distilled water. The data were reported in MPa and submitted to two-way ANOVA for randomized blocks, followed by Tukey's test (α = 0.05). Results The group irradiated with an Nd:YAG laser presented the lowest bond strength means, regardless of the μTBS test period (24 h or 6 months) (p < 0.05). No statistical difference was observed in the μTBS means among the groups CHX and NT (p > 0.05), nor did time influence bond strength (p > 0.05). Conclusion Immediate and long-term bond strength was negatively affected by Nd:YAG irradiation, a condition not observed with 2% CHX solution application.