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Staphylococcus aureus is one of the most common pathogens responsible for hospital-acquired infections. In this study, S. aureus was exposed to 13.56 MHz radiofrequency (RF) plasma generated by two different gases namely nitrogen and nitrogen–oxygen mixture and their sterilization efficacies were compared. Nitrogen plasma had a significant effect on sterilization due to generation of ultraviolet (UV) radiation. However, the addition of 2% oxygen showed enhanced effect on the sterilization of bacteria through nitric oxide (NO) emission and various reactive species. The presence of these reactive species was confirmed by optical emission spectroscopy (OES). Scanning electron microscopy (SEM) analysis was carried out to study the morphological changes of bacteria after plasma treatment. From the SEM results, it was observed that the bacterial cells treated by N2–O2 mixture plasma were severely damaged. As a result, a log10 reduction factor of 6 was achieved using N2–O2 plasma after 5 min treatment with 100 W RF power.