The present work is focused on the physicochemical characteristics, chemical composition, and some biological activities of Koelreuteria paniculata seed oil. The glyceride oil, obtained with a Soxhlet apparatus by extraction with hexane, was characterized by a relatively high oil content (over 20%), and it is defined as a non-drying oil (iodine value-44 gI2/100 g) with good oxidative stability (over 50 h). There were identified 11 fatty acids, 6 sterols, 3 tocopherols, and 6 phospholipids, as the last group was reported for the first time. The major components among them were-monounsaturated eicosenoic and oleic acids, β-sitosterol, β-tocopherol, and phosphatidylcholine. The in vitro tests demonstrated DNA protective activity and a lack of cytotoxicity of the oil, data that has been reported for the first time. The in vitro MTT test of the oil on HT-29 and PC3 cell lines did not indicate antitumor activity. The seed oil studied contains valuable bio-components, which have proven benefits for human health, and that is why it could be used in food, cosmetic, and pharmaceutical products.