The present work focusses on the chemical characterization and bioactive properties of Opuntia joconostle F.A.C. Weber ex Diguet and Opuntia matudae Scheinvar fruits. This research showed that xoconostle cv. Cuaresmeño (O. joconostle) and xoconostle cv. Rosa (O. matudae) are a good source of PUFAs and tocopherols. Moreover, both fruits revealed the presence of ten phenolic compounds (e.g., ferulic acid hexoside, quercetin-O-di-deoxyhexosyl-hexoside, and kaempferol-O-(di-deoxyhexosyl)-hexoside), as well as other organic acids (oxalic, malic, ascorbic and citric acids), and two betacyanins (betanin and isobetain). The hydroethanolic extracts of both fruits exhibited antioxidant activity, and inhibited the growth of several bacteria strains and of the yeast Candida albicans. As expected, xoconostle cv. Cuaresmeño was the fruit with highest antioxidant potential, since it was also the one that showed the highest content of bioactive compounds, with the exception of betacyanins. Overall, both fruits are revealed to be a good source of nutritive and bioactive compounds.