Six new pairs of isoquinoline alkaloid enantiomers, designated as yanhusanines A-F (<b>1</b>-<b>6</b>), were isolated from an aqueous extract of <i>Corydalis yanhusuo</i> tubers. The structures of these enantiomers were elucidated via physicochemical analysis and a variety of spectroscopic methods. All compounds were resolved into their enantiomers via chiral-phase HPLC, and their configurations were determined by DP4+ NMR calculation methods, specific rotations, and comparison of experimental and calculated ECD spectra. Compounds <b>1</b>-<b>6</b> bear a rare 9-methyl moiety, and compound <b>1</b> possesses a rare 1-oxa-6-azaspiro[4.5]decane core containing an <i>N</i>-CHO group. Compounds (+)-<b>2</b>, (-)-<b>2</b>, (+)-<b>4</b>, (-)-<b>4</b>, (+)-<b>5</b>, (-)-<b>5</b>, (+)-<b>6</b>, and (-)-<b>6</b> exhibited selective inhibitory activities against human carboxylesterase (hCE2), in the IC<sub>50</sub> value range of 2.0-13.2 μM.