The aim of this in vitro study was to evaluate the effect of different disinfection methods on the initial microtensile bond strength of a two-step, self-etch adhesive to dentin. Twenty mandibular molars were sectioned parallel to the occlusal plane to expose the mid-coronal dentin. All of the teeth were divided into four groups (n = 5 per group): (1) in group OZ, the dentin surfaces were exposed to ozone gas from the Ozonytron X delivery system (OzonyTron X-Bioozonix, Munich, Germany), (2) in group ND, the dentin surfaces were irradiated with an Nd:YAG laser (Pulsmaster 600 IQ, American Dental Technologies, U.S.), (3) in group CHX, the dentin surfaces were treated with a 2% chlorhexidine solution, and (4) in the control group, no treatment was applied. In all of the groups, the teeth were restored with Clearfil SE Bond (Kuraray, Tokyo, Japan) and Clearfil Majesty Posterior (Kuraray, Tokyo, Japan), according to the manufacturer's instructions. The teeth were sectioned perpendicular to the bonded surface (surface area of approximately 1 mm(2)). Thus, six to seven specimens were obtained from each tooth, and a total of 34 specimens were analyzed in each group. The specimens were attached to the microtensile test machine (Micro Tensile Tester, T-61010 K, Bisco, U.S.). The data was analyzed using the one-way analysis of variance (ANOVA) and Tukey test (p < 0.05). Fracture modes of each specimen were determined using a stereomicroscope (SZ-PT Olympus, Tokyo, Japan) and a scanning electron microscope (SEM). The lowest bond strength occurred in the OZ group. Significant differences were determined only between group OZ and the other groups (group ND, group CHX, and control group) (p < 0.05). In conclusion, although ozone decreased the microtensile bond strength of the self-etch adhesive system to dentin, the Nd:YAG laser and 2% chlorhexidine did not change the microtensile bond strength so in context of the present study it would appear that the Nd:YAG laser and 2% chlorhexidine may be used as pre-restorative sterilization procedures on the dentin prior to the application of a two-step, self-etch adhesive.