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<i>Eruca sativa</i> Mill. (<i>E. sativa</i>) leaves recently grabbed the attention of scientific communities around the world due to its potent bioactivity. Therefore, the present study investigates the metabolite profiling of the ethanolic crude extract of <i>E. sativa</i> leaves using high resolution-liquid chromatography-mass spectrometry (HR-LC/MS), including antibacterial, antioxidant and anticancer potential against human colorectal carcinoma cell lines. In addition, computer-aided analysis was performed for determining the pharmacokinetic properties and toxicity prediction of the identified compounds. Our results show that <i>E. sativa</i> contains several bioactive compounds, such as vitamins, fatty acids, alkaloids, flavonoids, terpenoids and phenols. Furthermore, the antibacterial assay of <i>E. sativa</i> extract showed inhibitory effects of the tested pathogenic bacterial strains. Moreover, the antioxidant activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide (H₂O₂) were found to be IC₅₀ = 66.16 μg/mL and 76.05 μg/mL, respectively. <i>E. sativa</i> also showed promising anticancer activity against both the colorectal cancer cells HCT-116 (IC₅₀ = 64.91 μg/mL) and Caco-2 (IC₅₀ = 83.98 μg/mL) in a dose/time dependent manner. The phytoconstituents identified showed promising pharmacokinetics properties, representing a valuable source for drug or nutraceutical development. These investigations will lead to the further exploration as well as development of <i>E. sativa</i>-based nutraceutical products.