Marine long chain omega-3 polyunsaturated fatty acids (ω3 FA) are involved in numerous cell responses and therefore vital for the mammal organism. Because of the attribution of immunomodulatory effects, a favorable impact on the inflammatory response in chronic wounds and cells involved in wound healing can be suspected. In the experimental setup Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) were investigated regarding their impact on metabolic activity, cell proliferation and migration of human keratinocytes (HaCaT) and newborn foreskin fibroblasts (CRL-2522). For simulation of the microenvironment of a chronic wound, human chronic wound fluid (CWF) was used in the experimental setup addressing the in vitro influence of DHA, EPA and CWF on regenerative processes. The results showed a significant increase in the metabolic activity of keratinocytes and fibroblasts after 72 h treatment with DHA and EPA. In contrast, treatment with ω3 FA had no significant positive effect on skin cell proliferation. Both ω3 FA had no influence on in vitro wound closure. CWF demonstrated significantly adverse effects, which ω3 FA were unable to mitigate. It can be concluded, that CWF exhibited the expected adverse effect on both skin cell types, especially inhibiting in vitro wound closure. ω3 FAs showed a slightly positive, yet rarely significant effect on human skin cells. Overall, the addition of DHA or EPA showed no relevant benefit for skin cells challenged with human chronic wound fluid (CWF), merely in combination with DHA an initial significant increase in cell metabolism (fibroblasts) and cell proliferation (keratinocytes) could be observed.