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Three new compounds (<b>1</b>, <b>11</b>, and <b>12</b>), together with 32 known ones, were isolated from the root bark of <i>Morus alba</i> L. using various chromatographic methods. The structures of the undescribed compounds were elucidated based on 1D, 2D NMR, and HRESIMS dataanalysis, while the known ones were identified by comparison of their spectroscopic data with those reported in the literature. All the isolates were evaluated for their cytotoxic activities against human gastric cancer HGC27 cells by CCK-8 assay. Among them, compounds <b>5</b>, <b>8</b>, <b>10</b>, and <b>30</b> exhibited cytotoxic activities on HGC27 cells with IC₅₀ values of 33.76 ± 2.64 μM, 28.94 ± 0.72 μM, 6.08 ± 0.34 μM, and 10.24 ± 0.89 μM, respectively. Furthermore, compound <b>10</b> was confirmed to reduce proliferation ability, increase apoptosis rate, and inhibit cell migration pathway by annexin V/PI double staining experiment, transwell experiment, and Western blot analysis.