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Nonthermal atmospheric-pressure plasmas provide new hope for medical application due to their potency in killing microorganisms. However, effectiveness and safety need evaluation. Here, the interaction of a nonthermal atmospheric-pressure plasma jet (APPJ) with human keratinocytes and cocultivated bacteria in an in vitro wound model is described. To evaluate the effects of the APPJ to 2-D cell cultures of human keratinocytes (HaCaT) in vitro, we modified the classical wound-healing assay (scratch assay) by adding an agarose overlay. In a further modification, the scratch was infected by 104 Staphylococcus epidermidis cells to mimic an infected wound. Models were then treated by 40-s APPJ, and cell performance was monitored. Within 24 h, scratch width in untreated models increased. However, in treated models, HaCaT cells were able to grow, and the scratch was beginning to close. Hence, it is possible to kill or to diminish the growth of a skin-associated microorganism within using nonthermal plasma in a cocultivation approach. Additionally, we observed the intra- and extracellular abundance of reactive oxygen species (ROS) generated by the APPJ using either dihydroethidium or 1, 1-diphenyl-2-picrylhydrazyl. In liquids, the reactivity of ROS with DPPH lasted for at least 10 min after 40-s APPJ treatment. Other than ROS activity, UV radiation may contribute to the inactivation of S. epidermidis. The intramicrobial Fenton reaction can be an explanation to the increased cell death of S. epidermidis compared to eukaryotic HaCaT keratinocytes. These data give evidence that nonthermal atmospheric-pressure plasma can act selectively regarding prokaryotic and eukaryotic cells.