In recent years, photobiomodulation (PBM) has been recognized as a physical therapy in wound management. Despite several published research papers, the mechanism underlying photobiomodulation is still not completely understood. The investigation about application of blue light to improve wound healing is a relatively new research area. Tests in selected patients evidenced a stimulation of the healing process in superficial and chronic wounds treated with a blue LED light emitting at 420 nm; a study in animal model pointed out a faster healing process in superficial wound, with an important role of fibroblasts and myofibroblasts. Here we present a study aiming at evidencing the effects of blue light on the proliferation and metabolism in fibroblasts and keratinocytes. Different light doses were used to treat the cells, evidencing inhibitory and stimulatory effects. Electrophysiology was used to investigate the effects on membrane currents, while Raman spectroscopy revealed the mitochondrial Cytochrome C (Cyt C) oxidase dependence on blue light irradiation. In conclusion, we observed that the blue LED light can be used to modulate the activity of human fibroblasts, and the effects in wound healing are particularly evident when studying the fibroblasts and keratinocytes co-cultures.