A perennial plant Nepeta multifida L. (syn. Schizonepeta multifida (L.) Briq.) is one of the most common species of the Lamiaceae family growing in Eastern Siberia and used in traditional oriental medicine. The chemical composition of N. multifida has not been sufficiently studied. Chromatographic separation of phenolic compounds of N. multifida leaves using column chromatography and preparative HPLC resulted in the isolation of sixteen compounds, including a new flavonoid identified as luteolin-7-O-(3′′,6′′-di-O-acetyl)-β-D-glucopyranoside. Known compounds were O-glycosides of luteolin and apigenin, rosmarinic acid, salvianolic acids A and B, and schizotenuin A. Quantitative analysis of N. multifida leaves by HPLC-UV assay showed the high content of rosmarinic acid (8.36–35.71 mg/g), luteolin-7-O-glucuronide (2.03–14.18 mg/g) and schizotenuin A (5.29–9.56 mg/g). The highest level of phenolic compounds was found in the flowering and fruiting phases. Using Ellman’s spectrophotometric method, it was found that N. multifida leaf extract and some compounds had antiacetylcholinesterase activity, and luteolin glycosides being the most active showed the level of concentration of half-maximal enzyme inhibition (IC50) 29.03–58.36 μg/mL. Thus, as a result of the present study, it was found that the leaves of N. multifida contain various groups of phenolic compounds capable of inhibiting the activity of acetylcholinesterase.