Green tea (Camellia sinensis) and Ginkgo biloba extracts in cosmetic formulations have been suggested to protect the skin against UV-induced damage and skin ageing. Thus, it is very important to assess the human skin penetration of their major flavonoids to verify if they penetrate and remain in the skin to exert their proposed effects. The aim of this study was to evaluate the human skin penetration of epigallocatechin-3-gallate (EGCG) and quercetin from green tea and G. biloba extracts vehiculated in cosmetic formulations. This study was conducted with fresh dermatomed human Caucasian skin from abdominal surgery mounted on static Franz diffusion cells. Skin samples were mounted between two diffusion half-cells and 10 mg/cm2 of formulations supplemented with 6% of green tea or G. biloba extract were applied on the skin surface. The receptor fluid was removed after 6 and 24 h and analyzed by high-performance liquid chromatography for the quantification of the flavonoids. The stratum corneum was removed by tape stripping and immersed in methanol and the epidermis was mechanically separated from the dermis and triturated in methanol to extract EGCG and quercetin. The results showed that the flavonoids under study penetrated into the skin, without reaching the receptor fluid. The majority of EGCG was quantified in the stratum corneum (0.87 μg/cm2), which was statistically higher than the EGCG concentrations found in viable epidermis (0.54 μg/cm2) and in the dermis (0.38 μg/cm2). The majority of quercetin was quantified in the viable epidermis (0.23 μg/cm2), which was statistically higher than the EGCG concentration found in the stratum corneum layer (0.17 μg/cm2). Finally, it can be concluded that EGCG and quercetin from green tea and G. biloba extracts vehiculated in cosmetic formulations presented good skin penetration and retention, which can favor their skin effects.