Inefficient autologous tissue recovery in diverse skin injuries increases the susceptibility of patients to infections caused by multiresistant microorganisms, resulting in a high mortality rate. Nonviral transfection is an attractive alternative for these patients, where genetically modified cells incorporated into skin substitutes could release additional antimicrobial agents into the native skin. In this work, we have modulated the conditions of using a nonviral system for transfection of primary human keratinocytes and fibroblasts, consisting of a polymer/plasmid DNA (pDNA) complex called polyplex and its effects on the expression of LL-37 antimicrobial peptide. Linear and branched polyethylenimine (PEI) polymers in different weight concentrations were varied for evaluating the formation and colloidal characteristics of the polyplexes. The PEI/pDNA polyplexes with 19 nitrogen/phosphate ratio are nanometric particles (400 and 250 nm with linear and branched PEI, respectively) exhibiting positive surface (+30 ± 2 mV). Both kinds of polyplexes allowed the expression of a reporter gene and increased the human cathelicidin antimicrobial peptide gene expression in transfected keratinocytes and fibroblasts; however, greater cytotoxicity was observed when polyplexes formed with branched PEI were used. Moreover, cell culture supernatants from transfected cells with linear PEI/pDNA polyplexes showed enhanced antimicrobial activity (decrease of bacterial growth in 95.8%) against astrain. The study of the PEI/pDNA polyplexes formation allowed us to develop an improved transfection strategy of skin cells, promoting the production of LL-37 antimicrobial peptide. In the future, this strategy could be used for the construction of skin substitutes to prevent, reduce, or eliminate bacterial infections. Impact statement The results of this study contribute to the understanding of the polyplexes system in the genetic modification of skin cells and its effects on the expression of the LL-37 antimicrobial peptide. In the future, three-dimensional skin substitutes built with these cells could be an efficient way to decrease bacterial growth and prevent the infections in skin wounds.