ABSTRACTIn response to the demand for N95 respirators by health care workers during the COVID-19 pandemic, we evaluated decontamination of N95 respirators using an aerosolized hydrogen peroxide (aHP) system. This system is designed to dispense a consistent atomized spray of aerosolized, 7% hydrogen peroxide (H2O2) solution over a treatment cycle. Multiple N95 respirator models were subjected to 10 or more cycles of respirator decontamination, with a select number periodically assessed for qualitative and quantitative fit testing. In parallel, we assessed the ability of aHP treatment to inactivate multiple viruses absorbed onto respirators, including phi6 bacteriophage, herpes simplex virus 1 (HSV-1), coxsackievirus B3 (CVB3), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For pathogens transmitted via respiratory droplets and aerosols, it is critical to address respirator safety for reuse. This study provided experimental validation of an aHP treatment process that decontaminates the respirators while maintaining N95 function. External National Institute for Occupational Safety & Health (NIOSH) certification verified respirator structural integrity and filtration efficiency after 10 rounds of aHP treatment. Virus inactivation by aHP was comparable to the decontamination of commercial spore-based biological indicators. These data demonstrate that the aHP process is effective, with successful fit-testing of respirators after multiple aHP cycles, effective decontamination of multiple virus species, including SARS-CoV-2, successful decontamination of bacterial spores, and filtration efficiency maintained at or greater than 95%. While this study did not include extended or clinical use of N95 respirators between aHP cycles, these data provide proof of concept for aHP decontamination of N95 respirators before reuse in a crisis-capacity scenario.IMPORTANCE The COVID-19 pandemic led to unprecedented pressure on health care and research facilities to provide personal protective equipment. The respiratory nature of the SARS-CoV2 pathogen makes respirator facepieces a critical protective measure to limit inhalation of this virus. While respirator facepieces were designed for single use and disposal, the pandemic increased overall demand for N95 respirators, and corresponding manufacturing and supply chain limitations necessitated the safe reuse of respirators when necessary. In this study, we repurposed an aerosolized hydrogen peroxide (aHP) system that is regularly utilized to decontaminate materials in a biosafety level 3 (BSL3) facility, to develop a method for decontamination of N95 respirators. Results from viral inactivation, biological indicators, respirator fit testing, and filtration efficiency testing all indicated that the process was effective at rendering N95 respirators safe for reuse. This proof-of-concept study establishes baseline data for future testing of aHP in crisis-capacity respirator-reuse scenarios.