In recent years, there has been considerable interest in bioactive peptides derived from food proteins, which might benefit human health. Pitaya and jiotilla are fruits with demonstrated biological functions in its stem, peel, and pulp. However, no studies have been conducted to evaluate the peptides. The aim of this study was to determine biofunctional properties (antioxidant, antihypertensive, and antidiabetic activities) of peptides from cacti seed protein. Antioxidant activity by ABTS, ACE inhibition activity, and α-amylase inhibition activity was carried out in the peptides. Based on the obtained results, hydrolyzed total protein isolate of pitaya showed high antioxidant activity (1328.40 ± 63.50 μM Trolox equivalents/mg protein). Moreover, hydrolyzed glutelin fraction of pitaya has a high α-amylase inhibition activity (76.74 ± 2.15%). On the other hand, hydrolyzed total protein isolate of jiotilla had a good ACE inhibition activity (47.64 ± 3.16). These results demonstrated that pitaya and jiotilla seeds protein hydrolysates had excellent in vitro nutraceutical potential.