Skin damage due to severe burns can compromise patient life. Current tissue engineering methods allow the generation of human skin substitutes for clinical use. However, this process is time-consuming, as the keratinocytes required to generate artificial skin have a low proliferation rate in culture. In this study, we evaluated the pro-proliferative effects of three natural biomolecules isolated from olive oil: phenolic extract (PE), DL-3,4-dihydroxyphenyl glycol (DHFG), and oleuropein (OLP), on cultured human skin keratinocytes. The results showed that PE and OLP increased the proliferation of immortalized human skin keratinocytes, especially at concentrations of 10 and 5 µg/mL, respectively, without altering cell viability. In contrast, DHFG did not produce a significant improvement in keratinocyte proliferation. In normal human skin keratinocytes obtained from skin biopsies, we found that PE, but not OLP, could increase the number of keratinocyte colonies and the area occupied by these cells. Furthermore, this effect was associated with increased KI-67 and Proliferating cell nuclear antigen (PCNA) gene expression. Thus, we propose that PE positively affects keratinocyte proliferation and could be used in culture protocols to improve bioartificial skin generation by tissue engineering.