Betacyanin (betanin), total phenolics, vitamin C and antioxidant capacity (by Trolox-equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays) were investigated in two differently colored cactus pear (Opuntia ficus-indica (L.) Mill.) genotypes, one with purple fruit and the other with orange fruit, from the Salento area, in Apulia (South Italy). In order to quantitate betanin in cactus pear fruit extracts (which is difficult by HPLC because of the presence of two isomers, betanin and isobetanin, and the lack of commercial standard with high purity), betanin was purified from Amaranthus retroflexus inflorescence, characterized by the presence of a single isomer. The purple cactus pear variety showed very high betanin content, with higher levels of phenolics, vitamin C, and antioxidant capacity (TEAC) than the orange variety. These findings confirm the potential for exploiting the autochthonous biodiversity of cactus pear fruits. In particular, the purple variety could be an interesting source of colored bioactive compounds which not only have coloring potential, but are also an excellent source of dietary antioxidant components which may have beneficial effects on consumers’ health.