Eucalyptus globulus is widely introduced and cultivated in Yunnan province. Its foliage is mainly used to extract eucalyptus oil, but the by-product eucalyptus residue has not been fully utilized. Based on the above reasons, in this study, we sought to explore the comprehensive utilization potential of eucalyptus resources. The total composition of eucalyptus residue was analyzed by ultra performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q/TOF MS), and the active components and nutrient components of eucalyptus leaf residue were determined by chemical methods and liquid phase techniques. Meanwhile, the antitumor activity of triterpenoids in eucalyptus leaves was evaluated by tetramethylazazole blue colorimetric assay (MTT). The results of qualitative analysis indicated that 55 compounds were identified from eucalyptus residue, including 28 phloroglucinols, 17 terpenoids, 3 flavonoids, 5 fatty acids, 1 amino acid and 2 polyphenols. Among them, the pentacyclic triterpenoids, in eucalyptus residue, were mainly oleanane type and urthane type. The results of quantitative determination indicated that the content of triterpenoid compounds was 2.84% in eucalyptus residue, which could be enhanced to 82% by silicone separation. The antitumor activity results showed that triterpenoid compounds have moderate inhibitory effects on human breast cancer cell MDA-MB-231, gastric adenocarcinoma cell SGC-7901 and cervical cancer cell Hela. The half maximal inhibitory concentration (IC50) was 50.67, 43.12 and 42.65 μg/mL, respectively. In this study, the triterpenoids from eucalyptus leaf residues were analyzed to reveal that the triterpenoids from eucalyptus leaf have antitumor effects and have potential to be developed as antitumor drugs.