Tyrosinase catalyzes the rate-limiting steps of melanin production, posing as an important target for treating skin pigmentation. This study investigates bioactive human tyrosinase inhibitors from Xanthium strumarium L. using a combined strategy of cell lysate, cell-based, and zebrafish assays. In this study, the methanol extract of Xanthium strumarium L. was identified as a potent inhibitor against tyrosinase in a cell lysate assay utilizing human MM418C1 melanoma cells. Subsequent phytochemical analysis resulted in the isolation of 11 natural products, including 4-hydroxybenzoic acid (4HB), three nucleotides, four caffeoylquinic acids and three alkaloids. Biological activity evaluation of isolated compounds suggested that 4HB was a potent inhibitor against tyrosinase with an IC50 value of 59.5 μg/mL. Further evaluations revealed that 4HB significantly reduced the melanin content by 40% at the concentration of 500 mg/mL in human MM418C1 melanoma cells. 4HB activity was finally confirmed in vivo, by the demonstration of 40% reduction in melanin production in live zebrafish at the concentration of 15.63 μg/mL.