One of the primary causes of human mortality is cancer. Herbal plants may offer new and enhanced anticancer compounds to combat cancer. The current study prepared, characterized, and evaluated a pumpkin seed aqueous extract (PAE) for its antioxidant and anticancer activities.The extraction yield from PAE was recorded 13 g/ 100g Defalted flour. The total phenol (TPCs) content of the PAE was 144.99 mg GAE g^-1 dry extract, while the total flavonoid (TFCs) content was 11.84 mg QE g^-1 dry extract. Twelve phenolic compounds (Gallic acid, Caffeic acid, Rutin, Coumaric acid, Vanillin, Naringenin, Querectin, Ellagic acid, 3.4-Dihydroxybenzoic acid, Keampferol, Ferulic acid, and Syringic acid) dominated in the chromatogram from the separation of PAE. It can be noted that the antioxidant activity of PAE increased gradually with increasing concentrations. When the concentration of extract was increased from 100 to 2000 µg/mL, the DPPH radical scavenging activity increased from 8.39% to 29.86%. The MTT assay revealed that the PAE inhibited the proliferation of the human cancer cell lines (HCT 116 and A-549) in a concentration-dependent manner. PAE had an IC₅₀ against HCT116 and A-549 (213.59, and 208.72 µg/mL, respectively). Therefore, PAE can be used as a source to produce pharmaceutical and medicinal products.