Three strawberry cultivars (Dover, Campineiro, and Oso Grande), grown in the same commercial plantation, were harvested at the ripe stage and stored at 6, 16 and 25 °C, for 6 days. During this period, chemical composition and antioxidant activity were evaluated. Results showed an increase in total soluble sugars, anthocyanin and vitamin C contents, indicating that a new biosynthesis had taken place during storage. Low temperature negatively affected anthocyanin and vitamin C accumulation, and positively affected soluble sugars, while flavonols, ellagic acid and total phenolic contents remained almost the same or even decreased at all temperatures. Despite differences in anthocyanin content between varieties and its increase during storage (higher with increasing temperature), there was no difference in the antioxidant activity between cultivars, which decreased after harvesting, independently of the temperature of storage. Variations in the proportion dehydroascorbic acid/ascorbic acid (DHA/AA) showed that there were differences between cultivars concerning adaptation of the fruit to low temperatures. The data obtained here indicate that cold storage is an effective way to maintain strawberry quality, but a compromise between sensorial and nutritional values can be achieved at 16 °C, for all the cultivars.