Rye (Secale cereale L.) is one of the most important cereal crops in Eastern and Northern Europe, showing better tolerance to environmental stress factors compared to wheat and triticale. Plant response to the crude oil-polluted soil depends on plant species, oil concentration, time of exposure, etc. The current study is aimed at investigating the growth, oxidative stress and the response of antioxidative system of two rye varieties (Krona and Valdai) cultivated on crude oil-contaminated soils at different concentrations (1.5, 3.0, 6.0, and 12.0%). Inhibition of rye growth was observed at crude oil concentrations of above 3% for above-ground plant parts and of above 1.5% for roots. A decrease in content of chlorophyll a and total chlorophylls in Krona variety was detected at 1.5% oil concentration in soil and in Valdai variety at 3% oil concentration. Compared with the control, the content of malondialdehyde was significantly increased in the Krona variety at 3% oil concentration and in Valdai variety at 6% oil concentration. The crude oil-induced oxidative stress was minimized in rye plants by the enhanced contents of low-molecular antioxidants (proline, non-protein thiols, ascorbic acid, phenolic compounds) and activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione peroxidase. The strongest positive correlation was detected between the content of malondialdehyde and contents of proline (r = 0.89–0.95, p ≤ 0.05) and phenolic compounds (r = 0.90–0.94, p ≤ 0.05) as well as superoxide dismutase activity (r = 0.81–0.90, p ≤ 0.05). Based on the results of a comprehensive analysis of growth and biochemical parameters and of the cluster analysis, Valdai variety proved to be more resistant to oil pollution. Due to this, Valdai variety is considered to be a promising rye variety for cultivation on moderately oil-polluted soils in order to decontaminate them. At the same time, it is necessary to conduct further studies aimed at investigating oil transformation processes in the soil-rye system, which would make it possible to determine the efficiency of using this cereal for soil remediation.