Fluorescent light energy (FLE) has been used to treat various injured tissues in a non-pharmacological and non-thermal fashion. It was applied to stimulate cell proliferation, accelerate healing in chronic and acute wounds, and reduce pain and inflammation. FLE has been shown to reduce pro-inflammatory cytokines while promoting an environment conducive to healing. A possible mechanism of action of FLE is linked to regulation of mitochondrial homeostasis. This work aims to investigate the effect of FLE on mitochondrial homeostasis in an in vitro model of inflammation. Confocal microscopy and gene expression profiling were performed on cultures of inflamed human dermal fibroblasts treated with either direct light from a multi-LED lamp, or FLE from either an amorphous gel or sheet hydrogel matrix. Assessment using confocal microscopy revealed mitochondrial fragmentation in inflamed cells, likely due to exposure to inflammatory cytokines, however, mitochondrial networks were restored to normal 24-h after treatment with FLE. Moreover, gene expression analysis found that treatment with FLE resulted in upregulation of uncoupling protein 1 () and carnitine palmitoyltransferase 1B () genes, which encode proteins favoring mitochondrial ATP production through oxidative phosphorylation and lipid β-oxidation, respectively. These observations demonstrate a beneficial effect of FLE on mitochondrial homeostasis in inflamed cells.