The purpose of this study was to evaluate the effects of sodium hypochlorite (5.25%), chlorhexidine (2%), and Glyde File Prep on the bond strengths of MTA-dentin in vitro. Standardized dentin disks were prepared and the central hole in each disk was filled with mineral trioxide aggregate (MTA). The specimens were randomly divided into four groups to be immersed in saline, 5.25% NaOCl, 2% chlorhexidine, and Glyde File Prep for 2 hours. The bond strengths of MTA-dentin were measured with a material testing system (MTS) and the fractured surfaces on the root walls were observed by scanning electron microscopy. Compared with the control group, the bond strengths were significantly lower in Glyde File Prep group (p < 0.05) and there was no significant difference in the chlorhexidine group or in the NaOCl group (p > 0.05). This study suggested that Glyde File Prep could negatively affect the bond strengths of MTA-dentin.