Although the pacemaker (PM) is a key cardiac implantable electrical device for life-threatening arrhythmias treatment, the related infection is a challenge. Thus, the aim of this study is to validate cold plasma as a potential technology for the disinfection of infected pacemakers. Fifty donated PMs were cleaned and sterilized before use and then infected with Staphylococcus aureus (S. aureus). Then, each experimental group was treated with cold plasma treatment for 1 min, 3 min, 5 min and 7 min, while the control group was immersed with sterilized water. Effectiveness of disinfection was evaluated by using CFU counting method and confocal laser scanning microscopy (CLSM). The physicochemical properties of water treated with cold plasma at different time were evaluated, including water temperature change and oxidation reduction potential (ORP). The major reactive species generated by the cold plasma equipment during cold plasma were analyzed with optical emission spectroscopy (OES). No live bacteria were detected with CFU counting method after 7 min of cold plasma treatment, which matches with the CLSM results. The ORP value of water and H2O2 concentration changed significantly after treating with cold plasma. Furthermore, reactive oxygen species (ROS) and reactive nitrogen species (RNS), especially NO, O (777 nm) and O (844 nm) were probably key inactivation agents in cold plasma treatment. These results indicate that cold plasma could be an effective technology for the disinfection of implantable devices.