The present study aimed to investigate the effect of Oenothera biennis sprout extract (OBS-E) on skin-function improvement in an in vitro system and to identify its pharmaceutically active components. OBS-E showed antioxidant ability in radical scavenging and reducing power assays, significantly inhibited matrix metalloproteinases-1 and -2, and increased the production of type I collagen, indicating its anti-wrinkle activity. Furthermore, OBS-E significantly increased the level of hyaluronic acid (HA) and the expression of moisturizing genes, such as hyaluronic acid synthase 2 (HAS2) and aquaporin 3 (AQP3), indicating it is effective in enhancing skin hydration. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) analyses showed that OBS-E contained high levels of polyphenolic acids, such as gallic acid and ellagic acid, in addition to flavonoid glycosides, such as luteolin 7-glucuronide and quercetin 3-glucuronide. Our results suggest that these major phytochemicals are likely to play crucial roles in the expression of antioxidant, anti-wrinkle, and moisturizing activities of OBS-E.