Diplotaxis species are edible and medicinal plants. This study investigates phenolic contents and biological activities in stems, leaves, and flowers of Diplotaxis harra and Diplotaxis simplex. Flowers exhibited the highest polyphenol content and D. harra was significantly more phenol-rich than D. simplex. Similarly, the highest antioxidant activities were found in D. harra flowers. Fractionation of D. harra extract revealed that 60% MeOH fraction showed the best antioxidant activities. Indeed, flower fractions were assayed for their activity against human pathogenic bacteria and the inhibitory percentages reached 100%. As regards fraction polarity, 60% and 80% MeOH fractions were the most active ones. Moreover, HPLC analysis showed that the main phenolic compound was cafeic acid for the two species, whereas p-Coumaric and 3,4-dimethoxybenzoic acids were identified only in D. simplex and sinapic acid was exclusive to D. harra. Overall, our findings indicate that Diplotaxis flowers could be used as a potent source of natural antioxidants and antibiotics.