Total phenolic content and antioxidant capacity of 11 commercial cultivars and 15 advanced breeding lines of spinach were determined over two growing seasons known to vary in biotic and abiotic stresses. Flavonoid composition and content of fall-grown commercial cultivars and advanced breeding lines were also determined. Over-winter spinach, which was planted in late fall and harvested in the spring, had much higher levels of total phenolics and antioxidant capacity than spinach planted in early fall and harvested in late fall, indicating that growing conditions, as well as biotic and abiotic stresses, influenced phenolic metabolism. Genotype also appeared to play an important role in affecting phenolic metabolism and antioxidant capacity in spinach. Advanced breeding lines of spinach, which show increased disease resistance, had higher levels of total phenolics, individual and total flavonoids, and antioxidant capacity than commercial cultivars. Our results indicate that plant breeders can select for increased phenolic content to increase antioxidant capacity in spinach cultivars, or the crops can be grown in different seasons or under certain stress conditions to elevate levels of antioxidants.