Honey is widely used for treating wounds, burns and ulcers, due to its antimicrobial and anti-inflammatory properties. Recently, it has been shown that it can also promote tissue repair. However, the mechanisms of action in the wound healing process are still far from full comprehension. In this work we propose an experimental methodology to investigate the potential role of honey as wound repair enhancer, by analyzing in vitro cell motility and proliferation, that are the main mechanisms involved in the wound repair. Our methodological approach is based on the well-assessed in vitro Wound Healing (WH) assay, coupled with Time-Lapse Microscopy (TLM) and image analysis techniques. Our methodology was applied to test the potential healing activities of several kinds of honeys and mixtures of selected honeys, on monolayers of HT1080 human fibrosarcoma cells. The honeys were also characterized for their antimicrobial activity. Overall, this work is focused on raising new therapeutic interest in the valuable natural product honey.