Casuarina equisetifolia L. (Casuarinaceae), an actinorhizal plant, exhibits mutualistic symbiosis with Frankia and promotes nitrogen fixation in root nodules. While the exchange of metabolites between host plant and microsymbiont is well understood in legume symbioses, the situation in the symbiosis between nitrogen-fixing Frankia and actinorhizal plants is less clear. In this study, a metabolomic approach was applied to root nodules of mature C. equisetifolia trees, leading to the identification of an undescribed taraxerane-type triterpenoid ester, 3-O-dihydrocoumaroyl β-taraxerol, along with twelve known compounds. An abundant component was tyramine with a content of 2.76 ± 0.315 mg/g FW in mature nodules. Tyramine specifically and abundantly accumulated in mature nitrogen-fixing nodules compared to senescent nodules, stems, leaves, and seeds. In addition, the potential function of tyramine was preliminarily examined and discussed.