Using a fractionation technique, four phytochemicals were isolated from Rhodiola crenulata extracts. These compounds were identified as 4'-hydroxyacetophenone (4-HAP), epicatechin-(4β,8)-epicatechin gallate (B2-3'-O-gallate), salidroside, and p-tyrosol using mass spectrometry and nuclear magnetic resonance spectroscopy. The inhibition of xanthine oxidase (XO) activity by these purified compounds was then evaluated and compared to that of a known XO inhibitor (allopurinol; IC50 = 12.21 ± 0.27 μM). Both 4-HAP and B2-3'-O-gallate showed an XO inhibitory effect, for which the half maximal inhibitory concentration (IC50) values were 15.62 ± 1.19 and 24.24 ± 1.80 μM, respectively. However, salidroside and p-tyrosol did not show significant inhibitory effects on XO at 30 μM. Furthermore, an inhibition kinetics study indicated that 4-HAP and B2-3'-O-gallate are mixed competitive inhibitors. The inhibition constants (Ki) of 4-HAP and B2-3'-O-gallate were 8.41 ± 1.03 and 6.16 ± 1.56 μM, respectively. These results suggest that 4-HAP and B2-3'-O-gallate are potent XO inhibitors.