Olive is one of the most important cultivated Mediterranean plants. In order to determine the differences in frost resistance of two, two-year-old olive cultivars (Olea europaea cv. Leccino and cv. Oblica) growing on different types of nutrient substrates (soil and coconut fibres), the trees were exposed to low temperature (-5 °C) in the dark. It was shown that low temperature caused an increase in H2O2 concentration, level of lipid peroxidation and carbonyl protein content in both cultivars and on both nutrient substrates, respectively. The CAT and APX activities significantly varied depending on the cultivar, the nutrient substrate type and the time of exposure to low temperature. Cv. Oblica and cv. Leccino growing on coconut fibres showed a better antioxidative response to low temperature probably due to the higher nitrogen and phosphorus concentration established in this type of nutrient substrate. That positive antioxidative response determined on coconut fibres was more pronounced in leaves of cv. Leccino.