In this work, we developed a device capable to generate a non-thermal plasma discharge inside a sealed bag. The aim of this study was to assess the effectiveness of the oxygen, nitrogen and argon plasma sterilization on Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis spores according to the NF EN 556 Norm. Moreover the bag integrity which is a critical key to maintain the sterile state of items after the end of the process was verified by Fourier Transform Infrared (FTIR) and X-ray Photoelectron Spectrometry (XPS) analyses. After plasma treatments, the bacterial counting showed a 6 log reduction of P. aeruginosa and S. aureus in 45 min and 120 min respectively whatever the gas used and a 4 log reduction of B. subtilis spores in 120 min with only oxygen plasma. These results were confirmed by Scanning Electron Microscopy (SEM) observations showing altered bacteria or spores and numerous debris. Taking into account the studied microorganisms, the oxygen plasma treatment showed the highest efficiency. FTIR and XPS analyses showed that this treatment induced no significant modification of the bags. To conclude this non-thermal plasma sterilization technique could be an opportunity to sterilize heat and chemical-sensitive medical devices and to preserve their sterile state after the end of the process.