Adlay (Coix lacryma-jobi L.), a Poaceae (Gramineae) plant, is cultivated in Thailand and other countries. It consists of numerous compounds, including phenolics, unsaturated fatty acids, triolein, coixenolide, and coixol, and possesses a variety of pharmacological activities. The adlay seed is the commonly used part of the plant in several foods and food supplements, but not in cosmetic products. The objectives of this study were to prepare the adlay seed extract and evaluate the total phenolic content. The antioxidant activity analyzed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and cell culture assays as well as the cytotoxicity assay were also investigated. The adlay seed extract was prepared by acidified ethanol using the maceration technique with the percent extraction yield of 7.72 ± 0.25% (w/w). The total phenolic content of the extract was 244.63 ± 1.19 mg gallic acid equivalent/g extract, and the concentration required to scavenge 50% of DPPH free radicals (IC₅₀) was 0.37 ± 0.00 mg/mL. The noncytotoxic concentrations of extract analyzed by sulforhodamine B assay were in the range of 0.0001-0.1 mg/mL. The antioxidant in cell culture assays showed the ability of the extract for the prevention of oxidative stress and treatment of oxidatively damaged cells in B16F10 melanoma cells. The study results suggested that adlay seed may potentially be used as an antioxidant ingredient in cosmetic preparations.