Objective To evaluate the sterilization efficacy of hysteroscopy instruments using ultraviolet C (UV-C) light at a wavelength of 259 nm in the Endoscopic Diagnostic Center of the National Institute of Perinatology. Methods Consecutive patients undergoing office hysteroscopy via the Bettochi vaginoscopy technique were included, excluding those with conditions such as viable intrauterine pregnancy or acute pelvic infection. Samples were collected from six designated sites of the hysteroscope, including the inner sheath, internal holes of the inner sheath, lens, graspers, scissors, and outer sheath. Initially, samples were taken after the first sterilization using a LAOKEN LK/MJG-150 Plasma Sterilizer (Chengdu, China). Next, samples were collected after the routine use of the hysteroscope in the office setting to confirm contamination. Subsequently, a new set of samples were taken after a 20-minute UV-C sterilization cycle with the EsteriUV device. Results The initial sterilization achieved a 96.73% sterilization rate, with ten samples testing positive for Staphylococcus coagulase-negative. Post-hysteroscopy, contamination increased significantly. Afterward, UV-C sterilization achieved a 96.08% sterilization rate, with 11 samples positive for Staphylococcus coagulase-negative and one for Streptococcus anginosus (p=0.66). No clinical infections were reported in any patient within one-month post-procedure. Conclusion UV-C light is a viable alternative for hysteroscopy instrument sterilization, demonstrating comparable efficacy to conventional methods. Further studies are recommended to optimize UV-C parameters for enhanced sterilization efficiency.