Physalis peruviana L. (P. peruviana) is an edible medicinal plant rich in bioactive phenolics. This study aimed to establish a hairy root (HR) culture of P. peruviana as a potential source for the synthesis of natural compounds. HRs were induced in P. peruviana using different Agrobacterium rhizogenes strains (R1601, C58C1, A4, and K599). Notably, K599 did not induce HR formation, whereas R1601, C58C1, and A4 yielded transformation frequencies of 57.78, 65.14, and 72.31%, respectively. Secondary metabolite production and antioxidant capacity were further examined in HRs induced using C58C1, R1601, and A4. It was found that A. rhizogenes R1601 induced the greatest increase (44% compared to that observed in the non-transformed culture). The methanolic extract of HRs induced by A. rhizogenes R1601 exhibited strong antioxidant capacity, with IC