Introduction: The maintenance of the aseptic chain associated with the knowledge and mastery of technique by the Endodontists are key factors for the success of endodontic treatment. Objective: This study aimed to evaluate, through a questionnaire, the care and cleaning, sterilization and storage methods of endodontic instruments, and the time of material disposal by endodontists from city of Caxias do Sul/ RS. Material and methods: Thirty-two endodontists participated in the study. The professionals answered a questionnaire with eight questions regarding biosecurity methods that they applied in their offices. Results: The cleaning process most adopted by most professionals was the use of brush associated or not with ultrasound. The disinfecting agent of choice by most respondents was the enzymatic detergent. All professionals performing the sterilization process by autoclave. Concerning to the disposal of manual endodontic instruments, fifteen participants reported to control the presence of twist or fracture and sixteen by the number of uses. For the rotary and reciprocating systems, most endodontists controlled the instrument replacement by the number of uses; with mean age of five uses for rotatory instruments. For the reciprocating system, different responses regarding the number of uses were reported. Conclusion: This study showed that the endodontists from Caxias do Sul/RS followed a satisfactory protocol for cleaning and sterilization of endodontic instruments. However, for the replacement of rotatory and reciprocating instruments, it was found that most professionals did not follow the manufacturers' recommendation.