Shiitake (Lentinula edodes) extract showed measurable anti-aging activity by inhibiting elastase by 51.99% and suppressing collagenase activity, targeting the enzymes that break down elastin and collagen in aging skin. The extract maintained over 90% viability in human keratinocytes up to 200 μg/mL and demonstrated moderate antioxidant capacity. A topical cream formulated at skin-compatible pH ~5.5 remained stable under accelerated aging conditions, suggesting shiitake is as a safe, enzyme-targeting ingredient for wrinkle support.