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This study was conducted to compare the effect of toothbrush sterilization using sodium bicarbonate and vinegar. The concentrations of the sodium bicarbonate were set to 1%, 3%, and 5% and the sterilization times were set to 10, 30, and 60 minutes. Additionally, the concentration of vinegar was set to 0.5%, 1%, and 2%, while the sterilization time was set to 5, 15, and 30 minutes. The test bacteria used were Streptococcus mutans, which cause cavities, and Staphylococcus aureus and Escherichia coli, which cause food poisoning and hygiene problems. The mean value and the significance of the number of bacteria remaining on the toothbrush were examined using the SPSS program and the optimal conditions were identified using Design expert. The results revealed that sodium bicarbonate effectively sterilized the samples when applied at 3% or 5% for 30 minutes, as did 1% or 2% vinegar applied for 30. However, the sterilization rate of sodium bicarbonate was 86–89%, which was more effective than the vinegar sterilization rate of 76–79%. In addition, considering the discomfort caused by the smell of vinegar, the most suitable method for application in child care facilities is sterilization in 3% or 5% solution of sodium bicarbonate for 30 minutes.