To investigate the action mechanism of ϵ-polylysine (ϵ-PL) against Escherichia coli (E. coli), a new field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) technology has been developed. The log phase E. coli cells were first incubated with ϵ-PL for 8 min, then the samples were directly added onto the silicon platelet and the copper grid, followed by a simple in situ fixation and freezing dehydration. FESEM and TEM were used to examine the ultrastructure changes in the bacterial envelope which was affected by ϵ-PL. various damages of E. coli cell envelope by ϵ-PL have demonstrated the detachment of outer membrane, the swelling of inner membrane, the apical burst of cells and the leakage of cytosol at a minimal inhibitory concentration (MIC) concentration. It also exhibited whole cell lysis at double MIC concentration. In summary, the new FESEM and TEM technology and appropriate sample preparation protocols have been found to be useful for investigating the biocidal activity of ϵ-PL against E. coli. SCANNING 35:412–417, 2013. © 2013 Wiley Periodicals, Inc.