Effective disinfection of healthcare devices is essential to prevent infections related to medical procedures and ensure patient safety. With the increasing use of 3D printing technologies in manufacturing these devices using polymers such as polylactic acid (PLA), there is a need for appropriate disinfection methods for such materials. However, traditional methods like chemical agents and sterilization by heat or radiation may have limitations when applied to PLA-printed instruments due to their porosity and complexity, which can harbor pathogenic microorganisms. These conventional disinfection methods may leave toxic residues on the parts, posing a risk to patient health, and may also affect the structural and functional integrity of medical devices due to exposure to high temperatures or radiation. The present study aims to investigate the feasibility and effectiveness of disinfecting PLA-printed instruments for biomedical applications using ozonated water. For the protocol, the printed instruments were experimentally contaminated with Staphylococcus aureus (S. aureus CCCD S013) and then subjected to the disinfection process in the hydrodynamic system using ozonated water (1.8 mg.L−1 (O3) for 10 min). The results showed a 3-log reduction in microorganism count, indicating a significant reduction in bacterial load, which is crucial for preventing infections associated with the use of these medical devices. These findings suggest that ozonated water may be a promising and sustainable alternative for disinfecting PLA instruments for biomedical applications, potentially contributing to the safety and quality of 3D-printed devices.