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Pitaya or dragon fruit comprises a group of exotic cacti species of the genus Hylocereus , with characteristic, eye-catching fruits. The present study analyzed the bioactive compounds (flavonoids, phenolic acids, total polyphenols and betacyanins) in different species (H. costaricensis, H. undatus and H. megalanthus) and varieties of fruits harvested in Israel. Direct comparisons of antioxidant and cytotoxic properties of the methanol and water extracts of the fruits, with the range of antioxidant tests (ABTS, CUPRAC, DPPH and FRAP) and also with normal (HaCaT and PNT2) and cancer (A375, WM793, Du145, HepG2, Caco-2, HT-29 and A549) cell lines was done. Advanced chemometric analysis was used to provide more information on the relationship between the fruits' compounds, varieties and biological activity of pitaya. Significant differences were observed between the tested pitaya samples for their chemical content and bioactivities. Red-skin pitaya fruits had the highest content of polyphenols and betacyanins and also had high antioxidant activity. The cytotoxic effect of H. costaricensis on cancer cells was greater than that of H. megalanthus and H. undatus , while none of the species was toxic to normal cells. The results showed that pitaya fruits have the potential to be included among the functional fruits. The species and even the variety of the pitaya fruit are of importance, as the different fruits varied significantly in their properties. [Display omitted] • Pitaya fruits of different species and varieties were compared. • Chemometric analysis indicated phytochemical content-activity relationships. • High antioxidant activity of red pitaya was observed. • Yellow pitaya showed cytotoxic, but not antioxidant activity. • Red pitaya was selectively cytotoxic to gastrointestinal cancer cells. [ABSTRACT FROM AUTHOR]