Ozonation is an innovative way to preserve high quality of seed during storage and it is an alternative to harmful organophosphorus agents. Disclosure of changes in the rapeseed oil under the influence of oxidative action of ozone on the seeds was the aim of the work and is presented for the first time. Two ozone concentrations and different length of ozonation were applied. The changes in tocopherols, phenolics, flavonoids contents, antiradical activity and oxidative stability were examined. The average vitamin E concentration was 33 mg per 100 g of oil before and 31.7 after ozonation of seeds. The average content of phenolic compounds was 14.3 mg of sinapic acid g−1 and 16.9 after the ozone treatment. Changes in the flavonoids content and antioxidant activity were observed too. Ozonation caused a decrease in the vitamin E concentration which resulted also in reducing the quenching of free radicals and reduction of oil induction time.