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Three types of extraction were used to obtain biologically active substances from the heartwood of <i>M. amurensis</i>: supercritical CO₂ extraction, maceration with EtOH, and maceration with MeOH. The supercritical extraction method proved to be the most effective type of extraction, giving the highest yield of biologically active substances. Several experimental conditions were investigated in the pressure range of 50–400 bar, with 2% of ethanol as co-solvent in the liquid phase at a temperature in the range of 31–70 °C. The most effective extraction conditions are: pressure of 100 bar and a temperature of 55 °C for <i>M. amurensis</i> heartwood. The heartwood of <i>M. amurensis</i> contains various polyphenolic compounds and compounds of other chemical groups with valuable biological activity. Tandem mass spectrometry (HPLC-ESI—ion trap) was applied to detect target analytes. High-accuracy mass spectrometric data were recorded on an ion trap equipped with an ESI source in the modes of negative and positive ions. The four-stage ion separation mode was implemented. Sixty-six different biologically active components have been identified in <i>M. amurensis</i> extracts. Twenty-two polyphenols were identified for the first time in the genus <i>Maackia</i>.