In this study, fatty acid compositions, physiochemical properties, and antioxidant activities of eight varieties of kiwifruit seed oil (KSO) in China were comparatively investigated. All varieties of KSO were rich in unsaturated fatty acid (UFA), especially linolenic acid. Different varieties of KSO showed subtle differences in physicochemical characteristics; e.g., density (0.88-0.091 mg/mL), refractive index (1.4803-1.4823), acid value (3.21-3.99 mg KOH/g oil), iodine value (177.22-202.75 g I/100 g oil), and peroxide value (7.23-14.75 mM/kg oil). Different varieties of KSO demonstrated different radical scavenging capacities for DPPH% (IC50 = 25.7-35.1 mg/mL), HO% (IC50 = 0.93-1.26 mg/mL), ORAC (1.26-1.99 mM Trolox/kg), and FRAP (3.30-132.30 mg Trolox/kg). Principal component analysis indicated the variety "Hongyang" to have a higher content of UFA and better antioxidant capacity than other varieties. Furthermore, comet assay verified that KSO can protect mice lymphocytes against oxidative DNA damage. Taken together, our findings provide evidence for the bioactivity of KSO as a potential dietary supplement.