Plasma-generated compounds (PGCs) such as plasma-processed air (PPA) or plasma-treated water (PTW) offer an increasingly important alternative for the treatment of microorganisms in hard-to-reach areas found in several industrial applications including the food industry. To this end, we studied the antimicrobial capacity of plasma-treated water on the vitality and biofilm formation of Listeria monocytogenes, a common food spoilage microorganism. Using a microwave plasma (MidiPLexc), 10 ml of deionized water was treated for 100 s, 300 s and 900 s (pretreatment time) and the bacterial biofilm was subsequently exposed to the PTW for 1 min, 3 min and 5 min (posttreatment time) for each pretreatment time separately. Colony-forming units (CFU), metabolic activity, and cell vitality were reduced for 4.7 log10, 47.9%, and 69.5%, respectively. Live/dead staining and fluorescence microscopy showed a positive correlation between treatment and incubation times and reduction in vitality. Atomic force microscopy indicated a change in the plasticity of the bacteria. These results suggest a promising antimicrobial impact of plasma-treated water on Listeria monocytogenes, which may lead to more targeted applications of plasma decontamination in the food industry in the future.