is a bacterium that causes various diseases in humans. Cold plasma is found to be an alternative to eliminate S. aureus. Most studies of cold plasma on S. aureus mainly focus on the physiochemical changes of the cells. So far, biological responses of S. aureus to cold plasma treatment under different treatment durations have not yet been evaluated. In this study, the results showed that the cold plasma was effective in eliminating S. aureus. At the initial exposure (1 min), the treated cells showed gene upregulations of stress proteins, antioxidants, nitrosative stress, and transporter proteins, but no significant change in other biological processes, such as cell membrane synthesis, DNA repairing, transcription, and translation. This indicated that the cells actively countered the damage from cold plasma. In contrast, during the prolonged treatment (3 and 5 min), biological processes related to central dogma were affected, including the DNA repairing mechanism, transcription, and translation. In addition, the majority of the genes related to cell membrane synthesis were downregulated, indicating that the treated cells could no longer sustain their cell integrity. In conclusion, this study elucidated how cold plasma inactivated S. aureus in a series of cold plasma exposures and highlighted the sequential transcriptomic responses of S. aureus.