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In recent years, new therapies, such as skin cell lines injections, have emerged to promote re-epithelialization of damaged areas such as chronic ulcers or to treat patients with severe burns. Antiseptics are commonly used during wound clinical management to avoid serious infections, but they may delay the healing process due to their apparent cytotoxicity to skin cells. The cytotoxicity of ethanol, chlorhexidine digluconate, sodium hypochlorite, povidone iodine and polyhexanide was evaluated in this in vitro study on human fibroblasts and keratinocytes. Treatments were applied to each cell type culture every 48 h for 14 days. To determine the cytotoxic of antiseptics, cell viability (Live/Dead®) and cell proliferation (AlamarBlue™) assays were performed on cell monolayers. Cell migration capacity was evaluated with a wound closure assay. Results showed how chlorhexidine digluconate and ethanol significantly reduced the viability of keratinocytes and inhibited cell migration. Povidone iodine followed by chlorhexidine digluconate significantly reduced fibroblast cell viability. Povidone iodine also inhibited cell migration. Sodium hypochlorite was the least detrimental to both cell types. If epithelial integrity is affected, the wound healing process may be altered, so the information gathered in this study may be useful in selecting the least aggressive antiseptic after treatment with new emerging therapies.