Objective: To investigate the chemical components and biological activities of avocado seed extract and fractions in order to determine the nutritional and pharmaceutical values of avocado seed. Methods: Various organic solvents were applied for extraction and fractionation of avocado seed. 1,1-diphenyl-2-picryl-hydrazyl, 2,2-azinobis-3-ethyl benzothiazoline-6-sulfonic acid, and DNA oxidation assays were applied for investigation of free radical scavenging activity. Nitric oxide production was measured by Griess reaction assay. Moreover, MTT assay was used to measure cancer cell growth inhibition. Results: The result indicated that the avocado seed contains (7.14 ± 0.40) g lipid/100 g, (1.67 ± 0.03) g protein/100 g, (54.0 ± 1.2) g carbohydrate/100 g, and (62.0 ± 2.3) mg gallic acid equivalent/g dried weight extract. Moreover, dichloromethane and ethyl acetate were revealed to be the highest free radical scavenging fractions with IC50 values of (48.0 ± 3.4) μg/mL (1,1-diphenyl-2-picryl-hydrazyl assay) and (22.0 ± 1.8) μg/mL (2,2-azinobis-3-ethyl benzothiazoline-6-sulfonic acid assay), respectively. Additionally, the avocado seed extract and fractions were able to protect against H2O2-induced DNA damage at the concentration of 100 μg/mL. On the other hand, the ethanol extract was effective in reducing nitric oxide production from lipopolysaccharide-stimulated RAW 264.7 macrophage cells without cytotoxic effect. Notably, the avocado seed significantly inhibited the proliferation of human lung A549 and human gastric BGC823 cancer cells at the concentration of 200 μg/mL, especially hexane (81 ± 3)% and dichloromethane (75 ± 2)% fractions. Conclusions: The results of the present study supported the avocado seed as potential by-product source for further development of health beneficial products.