Using a combination of chromatographic methods, one new flavonol glycoside, myricetin 3,7-di-O-α-L-rhamnopyranoside (1), and nine known compounds myricitrin (2), quercetin 3,7-di-O-α-L-rhamnopyranoside (3), quercitrin (4), desmanthin-1 (5), myricetin 3-O -(3″-O-galloyl)-α-L-rhamnopyranoside (6), (+)-catechin (7), benzyl O-β-D-glucopyranoside (8), 2-phenylethyl O-β-D-glucopyranoside (9), and corilagin (10) were isolated from the leaves of Ardisia splendens Pit. Based on an in vitro test against Coxsackie viruses A16 by SRB assay, only compounds 2, 5, and 10 exhibited activity against Coxsackie viruses A16 with IC50 values of 40.1, 32.2, and 30.5 μM, respectively. This result suggested that compounds 2, 5, and 10 might be potential agents for treating hand, foot and mouth diseases.