Background Ozone (O3) is an effective disinfectant agent that leaves no harmful residues. Due to the global health crisis caused by the COVID-19 pandemic, surgical masks are in high demand, with some needing to be reused in certain regions. This study aims to evaluate the effects of O3 for pathogen disinfection on reused surgical masks in various conditions. Methods O3 generators, a modified PZ 2–4 for Air (2000 mg O3/L) and a modified PZ 7 –2HO for Air (500 mg O3/L), were used together with 1.063 m3 (0.68 × 0.68 × 2.3 m) and 0.456 m3 (0.68 × 0.68 × 1.15 m) acrylic boxes as well as a room-sized 56 m3 (4 × 4 × 3.5 m) box to provide 3 conditions for the disinfection of masks contaminated with enveloped RNA virus (105 FFU/mL), bacteria (103 CFU/mL) and fungi (102 spores/mL). Results The virucidal effects were 82.99% and 81.70% after 15 min of treatment with 2000 mg/L O3 at 1.063 m3 and 500 mg/L O3 at 0.456 m3, respectively. The viral killing effect was increased over time and reached more than 95% after 2 h of incubation in both conditions. By using 2000 mg/L O3 in a 1.063 m3 box, the growth of bacteria and fungi was found to be completely inhibited on surgical masks after 30 min and 2 h of treatment, respectively. Using a lower-dose O3 generator at 500 mg O3/L in 0.456 m3 provided lower efficiency, although the difference was not significant. Using O3 at 2000 mg O3/L or 500 mg O3/L in a 56 m3 room is efficient for the disinfection of all pathogens on the surface of reused surgical masks. Conclusions This study provided the conditions for using O3 (500–2000 mg/L) to reduce pathogens and disinfect contaminated surgical masks, which might be applied to reduce the inappropriate usage of reused surgical masks.