Olive tree growth and reproduction are severely affected by temperature extremes, compromising fruit yield. In that aspect, the olive varieties “Koroneiki” and “Mastoidis” were employed in a mild cold stress experiment, imitating night frost incidents to assess their biochemical, physiological and reproductive functions in relation to genotype. The physiological performance of the stressed plants was not significantly altered, suggesting that both cultivars were well adapted to mild cold night stress. The biochemical response of the plants, regarding antioxidant enzymes, H2O2 and TBARS accumulation, confirmed that both cultivars could cope with the stress applied. The mRNA levels of the PPO gene, which participates in hydroxytyrosol biosynthesis and plant defense, were elevated after 24-h stress at 0 °C, in both cultivars with “Mastoidis” plants exhibiting higher levels for a longer period. Three more genes involved in hydroxytyrosol biosynthesis upregulated their expression levels as a response to cold stress. The numerous plant phenology aspects measured reinforced the conclusion that both cultivars responded to the stress applied. The results of the present study may contribute to better understanding olive tree adaptive responses to low temperature events, an abiotic stress condition that is often present in an open plantation, thus assisting farmers on breeding and cultivar selection.