The objective of this study was to determine the effect of oxidative stress caused by exposure to ozone on the activity of 17-β-hydroxysteroid dehydrogenase and alkaline phosphatase, and testosterone concentrations in male rats, and to investigate the possible protective effect of easily available antioxidants such as vitamins E and C. The experiment was conducted on adult Wistar-Hannover rats. One group of animals was exposed to ozone without vitamin cover, and the remaining animals were administered vitamins E and C in various combinations and doses. Ozone exposure in the group of rats not receiving vitamin injections caused oxidative stress manifested by elevated MDA concentrations in the blood plasma and testicular tissue. An increase in MDA levels was also observed in the group of animals administered vitamins, excluding the animals receiving low- and average-dose combinations of vitamins E and C. A drop in the activity of 17-β-hydroxysteroid dehydrogenase was reported in animals exposed to ozone, but this effect was not noted in the groups exposed to ozone and receiving vitamins. The lowest blood testosterone levels were observed in rats exposed to ozone and in the groups receiving low- and average-dose combinations of vitamins E and C.