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The phytochemical composition, in vitro antioxidant and antimicrobial activities, cytotoxicity and antigenotoxicity of fruit extracts of Opuntia dillenii were studied. The phytochemical composition was evaluated using HPLC, GC-MS and UV–Vis spectrophotometry. Spectrophotometrical methods were used to estimate the antioxidant potential. Antimicrobial activity was determined using a microdilution method. The cytotoxic effects of the extracts were evaluated using the MTT assay. In vitro DNA-protective activity against hydroxyl radical-induced DNA damage was also determined. The results showed that polar extracts of O. dillenii had a significant amount of phenolic compounds, including flavonoids, whereas non-polar extracts had mostly terpenoids and fatty acid derivatives. Moreover, several extracts showed good antioxidant and antimicrobial activities, with low cytotoxicity and significant DNA-protective effects. These results showed that the extracts of O. dillenii have promising bioactivity and further studies on the potential application in different areas of food and health might be beneficial. • Opuntia dillenii extracts had complex phytochemical composition. • O. dillenii extracts showed promising antioxidant and antimicrobial properties. • Some of O. dillenii extracts had cytotoxicity with LoVo and MCF-7 cells. • O. dillenii extracts protected DNA from hydroxyl radical-induced damage. [ABSTRACT FROM AUTHOR]