Opuntia species are well-known for their use in folk medicine and richness in many bioactive compounds. This study aims to realize a taxonomic study and to evaluate the polyphenols content and antioxidant potential of edible parts of cultivated and wild Opuntia sp. fruits, using different in-vitro bioassays. The phylogenetic analysis confirmed the assignment of the samples to Opuntia genera. The Opuntia fruit fractions (seeds, pulp, and entire fruit) exhibited different amounts of polyphenols, with the highest values recorded for the wild species, and particularly their pulp (1140.86 mg GAE/100 g DM, and 155.62 QE/100 g DM for total phenolic and flavonoid compounds, respectively). Among the antioxidant activities, wild pulp exhibited the greatest antioxidant potential with a high radical scavenging activity (72.34% and 92.12% for hydrogen peroxide and hydroxyl radicals, respectively). The best nitric oxide scavenging activity was found for cultivated fruit fraction, with 55.22%. The statistical analysis also confirmed a significant correlation between the antioxidant activities and the phenolic compounds and flavonoids (>0.90, p ≤ 0.001) in all Opuntia extracts. Finally, both Opuntia fruits demonstrated a good antioxidant potential, enhancing the interest of this species as a non-pharmacological approach in a wide variety of disorders and diseases associated with oxidative stress, and paving the way to Opuntia sp. economic valorization.