Abstract Polyphenols from flesh and peels of red pitaya (Hylocereus polyrhizus) fruit were quantified by employing an extract sub-fractionation procedure applied for the first time to this fruit. Higher polyphenolic contents than those from the literature were measured by using analogous spectroscopic techniques to those reported in previous works. Betacyanin fractions exhibited the highest reducing and radical-scavenging capacities among the extracts and fractions tested by FRAP and DPPH assays, respectively. Finally, polyphenolic fractions showed a broad antimicrobial spectrum by inhibiting the growth of all of food-borne pathogens tested, while the non-fractionated extracts revealed a very low or no activity. Results indicated flesh as a good source of antioxidants with healthy benefits for human diet and peels as a valuable manufacture by-product to be exploited for the formulation of nutraceuticals and food applications.