Quercus acuta Thunb. (Q. acuta) is a subtropical, evergreen, broad-leaved tree with excellent carbonsequestration capacity and ecological adaptability. However, the phytochemical composition of fruits, particularly phenolic marker compounds, has not been sufficiently investigated. In this study, the biological activities of water and ethanol extracts of Q. acuta fruits were evaluated, and a high-performance liquid chromatography with diode array detection (HPLC-DAD)-based method for the quantification of marker compounds was established and validated. MTT assays showed no cytotoxicity at concentrations of 5–20 μg/mL, and the water extract significantly inhibited UVB-induced MMP-1 expression in HaCaT keratinocytes. Gallic acid and ellagic acid in Q. acuta fruits were confirmed as marker compounds, and the validated method demonstrated excellent linearity (R² > 0.999), recovery, and precision (RSD < 2.0%). Quantitative analysis revealed that the water extract contained higher levels of phenolic compounds compared with the 70% and 100% ethanol extracts. These findings support the stability and biological efficacy of phenolic marker compounds in aqueous environments and suggest the potential industrial applications of Q. acuta fruit extracts.