BACKGROUND The periapical area is healed through disinfection of root canal system and reduction of microbial infection after root canal. OBJECTIVE To assess the knowledge of dental practitioners about decontamination during root canal treatment and the techniques used in the government and private sectors of Jeddah, Saudi Arabia. METHODS 103 dental practitioners and interns from private and governmental sectors performing root canal treatment were included. To extract information, a questionnaire assessing knowledge and preferred techniques used in decontamination during root canals treatment was distributed. The association of variables was investigated using chi-square tests. RESULTS The findings reflected that 82.5% of subjects used rubber dam for isolation with significantly more practitioner in the governmental (95.2%) as compared to the private sector (27.8%). Chelating agents were used by 13.3% of the practitioners in government sector and 1% practitioners in private sector (1%). The most commonly used irrigant was sodium hypochlorite. Calcium hydroxide was used more frequently in the governmental sector (29.8%) than in the private sector (11.8%), as intracanal medicament. Mechanical irrigation devices were used by 2.4% of practitioners in the governmental sector only. CONCLUSIONS There is a significant difference in practicing root canal disinfection techniques among dentists in governmental and private sectors but no difference in degree of knowledge.