Aims The main objective of this study was to determine the inactivation efficacy of dielectric barrier discharge atmospheric cold plasma (DBD-ACP) generated inside a sealed package for Escherichia coli ATCC 25922. Methods and Results A plasma discharge was generated between two circular aluminium electrodes at 40kV. E.coli suspensions (107CFUml1) in either maximum recovery diluent (MRD) or phosphate buffered saline (PBS) were treated in a 96-well microtitre plate inside a sealed package. The effects of treatment time, post-treatment storage time, either direct or indirect samples exposure to the plasma discharge and suspension media were studied. Regardless of the media tested, 20s of direct and 45s of indirect plasma treatment resulted in complete bacterial inactivation (7logCFUml1). At the lower plasma treatment times (1030s) investigated, the effects of suspension media and mode of exposure on the inactivation efficacy were evident. The inactivation efficacy was also influenced by the post-treatment storage time. Conclusions It was demonstrated that the novel DBD-ACP can inactivate high concentrations of E.coli suspended in liquids within sealed packages in seconds. Significance and impact of the Study A key advantage of this in-package nonthermal novel disinfection approach is the elimination of post-processing contamination.