The cytotoxic activity of petroleum ether extract of the leaves of Cassia roxburghii Linn. against HCT-116 and MCF-7 cell lines resulted with IC50=34.9 and 38.04 μg/ml, respectively, while against HepG-2 showed no activity. A bioassay guided-fractionation approach was conducted to isolate and identify the active cytotoxic principles. Further chromatographic separation and purification of the petroleum ether extract resulted in the isolation of two anthraquinones identified as aloe-emodin acetate and aloe-emodin, along with stigmasterol, β-sitosterol and palmitic acid. The structure elucidation of isolated compounds was performend using 1D, 2D-NMR and HR-MS. Furthermore, the cytotoxicity of aloe-emodin acetate and aloe-emodin were evaluated and resulted with IC50=153.30 and 70.02 μg/ml against HCT-116 and with 93.20 and 53.20 μg/ml against MCF-7, respectively, while against HepG-2 showed no activity. Moreover, the antiviral activity of the two isolated anthraquinones was tested against influenza virus-A, and resulted with IC50=10.23 as well as 2.00 and with CC50=1.32 and 0.47 μg/ml, respectively.