The increasing global demand for diverse and health-promoting foods has led to the expansion of tropical fruit cultivation beyond their native regions, notably into the Mediterranean area. This shift necessitates a deeper understanding of their phytochemical profiles, as environmental factors in new cultivation contexts can significantly influence the biosynthesis of their bioactive compounds. In this study, we explored the phytochemical and antioxidant properties of pitaya fruit, focusing on chemical fractionation and the link between its bioactive components and functional benefits. Using HPLC-DAD-MS/MS, key compounds contributing to the fruit's antioxidant effects were identified. The total polyphenol content (TPC) in the original pitaya extract was 670.57 ± 15.98 mg 100 g of FW. Both in vitro (ABTS, DPPH, and FRAP) and the cellular antioxidant activity (CAA) assay demonstrated a dose-dependent prevention of oxidative damage, with 50% inhibition (IC) observed at 2.365 ± 0.072 µg/mL for ABTS, 20.797 ± 0.950 µg/mL for DPPH, and 7.28 ± 0.050 µg/mL for the CAA assay. Additionally, the DNA protection assay revealed that pitaya extract significantly reduced oxidative damage caused by HO and UV exposure, with a significant reduction in damage at the lowest tested concentration (100 µg/mL). Following chromatographic fractionation via flash chromatography, the fraction enriched in betacyanin (F2), and betaxanthine (F3) exhibited the strongest antioxidant effects on in solution-based assays. Interestingly, the flavonoid-rich fractions (F5 and F7), despite showing lower activity in these solution assays, provided nearly complete protection in cell-based biological models, raising the possibility that additional, and/or complementary antioxidant mechanisms could be involved. These findings underscore the potential of pitaya fruit as a valuable natural source of antioxidants for nutraceutical and therapeutic applications aimed at counteracting oxidative stress and related pathologies. PRACTICAL APPLICATIONS: 1. Betalains, in the F2 fraction, play a key role in the antioxidant potential of pitaya fruit. 2. DPPH and ABTS assays rank pitaya among the fruits with high antioxidant potential. 3. Pitaya fruit extract preserves up to 90% of DNA integrity against oxidative insults. 4. 80% of detected bioactive compounds were betalains and 20% were flavonoids.