Producing temperate-zone fruit crops in subtropical environments requires alterations in fertilizer application and rates. Nitrogen (N) is a critical mineral nutrient required in high amounts by the tree; however, it is often over- or under-applied for optimal fruit quality and can affect the phytochemical composition of fruits. The effects of different N fertilizer rates and harvest date on total phenolic content, total flavonoid content, total anthocyanins, total antioxidant capacity, total soluble solids, titratable acidity, and organic acids (citric and malic acid) of two subtropical peach (Prunus persica) cultivars, TropicBeauty and UFSharp, were investigated. N rate did not affect total soluble solids in ‘TropicBeauty’, although total soluble solids decreased as N rate increased in ‘UFSharp’. Titratable acidity and organic acid content was significantly higher in ‘UFSharp’ as compared with ‘TropicBeauty’, although there was no effect of N rate on titratable acidity. An overall increase in phenolic content, flavonoid content, anthocyanins, and antioxidant capacity were observed with decreasing N rates in both subtropical peach cultivars. A stronger genotype × N treatment interaction was observed for ‘TropicBeauty’ for phenolic content, flavonoid content, and antioxidant capacity than for ‘UFSharp’. In ‘TropicBeauty’, among the treatments with no N and highest N, an almost 100% increase in phenolic content, 200% increase in flavonoid content, 50% increase in anthocyanin content, and 80% increase in antioxidant activity was observed. A positive correlation among phenolic content, flavonoid content, and antioxidant capacity was observed in both ‘TropicBeauty’ and ‘UFSharp’. Late harvest date decreased phenolic content in ‘TropicBeauty’, ranging from 6% to 32% among different N treatments. Late harvest increased anthocyanin content as compared with fruit that were harvested on early dates. The results suggest that subtropical peach phytochemical composition can be affected by different cultivars and tree age, and can be manipulated with cultural practices like N fertilization and harvest time to produce fruit with altered or desired nutritional composition for consumers.