Objective: This cross-sectional observational study was designed to assess the biosafety conducts adopted by orthodontists, and possible differences regarding training time. Methods: Both the application of methods for sterilization/disinfection of instruments and materials, and the use of personal protective equipment (PPE) were collected through questionnaires via e-mail. Results: The questionnaires were answered by 90 orthodontists with a mean age of 37.19 ± 9.08 years and mean training time of 13.52 ± 6.84 years. Regarding orthodontic pliers, 63.23% use an autoclave, except 1 who does not perform any procedure. All participants use autoclave to sterilize instruments, and 95.6% of respondents perform cleaning with chemicals prior to sterilization. Most of them (65.56%) use an autoclave to sterilize orthodontic bands, with some still associating disinfection methods, while few (18.89%) do nothing at all. There was a high incidence of the answer “nothing” for the methods used for elastic, accessories, bandages, metal springs, and arches. All respondents use mask and gloves in attendance, 78.92% use aprons, 58.92% use protective goggles, and 50.01% use cap. Training time significantly influenced (p = 0.003) only the use of glutaraldehyde for sterilization/disinfection of pliers. Conclusions: The sterilization and cleaning of pliers, instruments, and bands, besides the use of PPE, received more uniform and positive responses, while other items suggest disagreements and possible failures. Only orthodontists trained for more than 13 years choose using glutaraldehyde for pliers sterilization/disinfection, the only adopted method with a significant difference in relation to training time.