In this study, we investigated the effect of enhanced collagen synthesis by Mychonastes sp. 249 (a type of freshwater microalgae) extract (MSE) on skin regeneration. The effect of MSE on human dermal fibroblast (HDF) migration was studied to confirm its skin regenerative effect, and at 1.0 mg/mL MSE increased HDF cell migration by 21.2%. To study the mechanism responsible, the mRNA levels of MMP-1 (matrix metalloproteinase-1), COL1A1 (collagen type I alpha 1), and SMAD-3 (suppressor of mothers against decapentaplegic-3) were evaluated by RT-PCR. The results showed that in the concentration range 0.0 to 1.0 mg/mL, MSE dose-dependently affected mRNA expressions in HDF cells (P<0.05). LC-MS/MS analysis identified eriodictyol (a flavonoid) as the main component of MSE. These results suggest that MSE could be used as a skin regeneration agent that inhibits collagen decomposition in HDF cells.