Introduction: Teeth intended for use as autogenous augmentative material may carry microbiological contamination, which can compromise the safety of regenerative procedures in the oral cavity. Therefore, effective disinfection protocols are crucial to ensure the microbiological purity of dentin-derived graft materials. Objective: This study aimed to evaluate the effectiveness of a 30% alkaline ethanol solution containing 0.5 M sodium hydroxide in eliminating microorganisms from dentin material intended for autogenous augmentation. Materials and Methods: The study included 44 extracted teeth that were processed using the Smart Dentin Grinder procedure. The presence of microorganisms was analysed using standard microbiological methods before and after treatment with the disinfectant solution. Additionally, the potential association between tooth eruption status and the effectiveness of the disinfection process was evaluated using Fisher’s exact test, with odds ratios calculated using the Haldane–Anscombe correction to address zero cell counts. Results: Processing dentin in a 30% alkaline ethyl alcohol solution containing 0.5 M sodium hydroxide is an effective method for eliminating microorganisms, thereby rendering the material completely microbiologically pure. Conclusions: The dentine processing procedure used appears to ensure the production of autogenous material free from microbiological contamination, indicating its potential safety in clinical applications.