Breast cancer is one of the most prevalent cancers in the world and is also the leading cause of cancer death in women. The use of bioactive compounds of functional foods contributes to reduce the risk of chronic diseases, such as cancer and vascular disorders. In this study, we evaluated the antioxidant potential and the influence of pitaya extract (PE) on cell viability, colony formation, cell cycle, apoptosis, and expression of BRCA1, BRCA2, PRAB, and Erα in breast cancer cell lines (MCF-7 and MDA-MB-435). PE showed high antioxidant activity and high values of anthocyanins (74.65 ± 2.18). We observed a selective decrease in cell proliferation caused by PE in MCF-7 (ER+) cell line. Cell cycle analysis revealed that PE induced an increase in G0/G1 phase followed by a decrease in G2/M phase. Also, PE induced apoptosis in MCF-7 (ER+) cell line and suppressed BRCA1, BRCA2, PRAB, and Erα gene expression. Finally, we also demonstrate that no effect was observed with MDA-MB-435 cells (ER−) after PE treatment. Taken together, the present study suggests that pitaya may have a protective effect against breast cancer.