Background: Orthodontic auxiliaries are frequently reused and may act as potential sources of cross-infection if not adequately sterilized. Limited comparative data exist regarding the effectiveness of commonly used sterilization methods for orthodontic materials. Aim: To evaluate and compare the effectiveness of different sterilization and disinfection methods in eliminating microbial contamination from selected orthodontic materials. Materials and Methods: This in-vitro microbiological study evaluated 48 orthodontic samples including NiTi closed coil springs, pre-formed molar bands with buccal tubes, and Class II elastics. Samples were divided into five groups: control, 70% ethanol, 2% glutaraldehyde, ultraviolet (UV) irradiation, and autoclaving. Following sterilization, all specimens were cultured on tryptic soy agar and incubated at 37°C for 72 hours. Microbial growth was assessed visually. Statistical analysis was performed using chi-square test. Results: All unsterilized samples demonstrated microbial growth. No microbial growth was observed in any samples treated with 70% ethanol, 2% glutaraldehyde, UV irradiation, or autoclaving. Statistically significant differences were observed between control and treated groups (Chi-square = 8.778, p = 0.003). Conclusion: All evaluated sterilization methods were effective in eliminating microbial contamination from orthodontic materials. Autoclaving and glutaraldehyde immersion are recommended as primary methods, while UV irradiation and ethanol can serve as adjunctive alternatives.