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<i>Ajuga bracteosa</i> Wall. ex Benth. is an endangered medicinal herb traditionally used against different ailments. The present study aimed to create new insight into the fundamental mechanisms of genetic transformation and the biological activities of this plant. We transformed the <i>A. bracteosa</i> plant with <i>rol</i> genes of <i>Agrobacterium rhizogenes</i> and raised the regenerants from the hairy roots. These transgenic regenerants were screened for in vitro antioxidant activities, a range of in vivo assays, elemental analysis, polyphenol content, and different phytochemicals found through HPLC. Among 18 polyphenolic standards, kaempferol was most abundant in all transgenic lines. Furthermore, transgenic line 3 (ABRL3) showed maximum phenolics and flavonoids content among all tested plant extracts. ABRL3 also demonstrated the highest total antioxidant capacity (8.16 ± 1 μg AAE/mg), total reducing power, (6.60 ± 1.17 μg AAE/mg), DPPH activity (IC50 = 59.5 ± 0.8 μg/mL), hydroxyl ion scavenging (IC50 = 122.5 ± 0.90 μg/mL), and iron-chelating power (IC50 = 154.8 ± 2 μg/mL). Moreover, transformed plant extracts produced significant analgesic, anti-inflammatory, anticoagulant, and antidepressant activities in BALB/c mice models. In conclusion, transgenic regenerants of <i>A. bracteosa</i> pose better antioxidant and pharmacological properties under the effect of <i>rol</i> genes as compared to wild-type plants.