The aims of this study were to identify microorganisms on the surface of gutta-percha (GP) cones using a polymerase chain reaction (PCR) and to evaluate the sterilization effect of 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and ChloraPrep on these bacteria. Alterations of the surface texture and physical properties of GP cone after chemical sterilization were compared. A total of 150 GP cones were randomly selected in endodontic clinics, and a PCR assay was performed to identify the contaminating microorganisms. After inoculation with the same microorganisms followed by drying for 1 day, the GP cones were immersed in the three chemical disinfectants. The sterilization effects were assessed by measuring the turbidity and through subculturing. The changes in surface texture were observed by scanning electron microscopy, and Instron 5500 (Instron Corp, Canton, MA) was used to measure the tensile strength and elongation rate of the GP cones after chemical sterilization. Statistical analysis was performed. Results indicate that 19.4% of GP cones from the clinic were contaminated, and all the species belonged to the genus Staphylococcus. Three chemical disinfectants were all effective in the rapid disinfection of GP cones against Staphylococcus spp, and 1-minute immersion of the GP cones was adequate for disinfection. Microphotographs of the NaOCl-soaked cone showed a cluster of cuboidal crystals. The tensile strength between the NaOCl- and CHX-soaked groups was significantly different (p<0.05), but there was no significant difference between the NaOCl- and ChloraPrep-soaked groups (p>0.05). All disinfectants significantly increased the elongation rate of the GP cones compared with fresh GP cones (p<0.05), especially in the ChloraPrep (Medi-flex, Leawood, KS). These results show that the three chemical disinfectants are effective agents for the rapid sterilization of GP cones. However, further research will be needed to determine the clinical relevance of the changes in physical properties.