Fagonia indica (L.) is an important medicinal plant with multitude of therapeutic potentials. Such application has been attributed to the presence of various pharmacological important phytochemicals. However, the inadequate biosynthesis of such metabolites in intact plants has hampered scalable production. Thus, herein, we have established an in vitro based elicitation strategy to enhance such metabolites in callus culture of F. indica. Cultures were exposed to various doses of UV radiation (UV-C) and grown in different photoperiod regimes and their impact was evaluated on biomass accumulation, biosynthesis of phytochemicals along antioxidant expression. Cultures grown under photoperiod (16L/8D h) after exposure to UV-C (5.4 kJ/m) accumulated optimal biomass (438.3 g/L FW; 16.4 g/L DW), phenolics contents (TPC: 11.8 μgGAE/mg) and flavonoids contents (TFC: 4.05 μgQE/mg). Similarly, HPLC quantification revealed that total production (6.967 μg/mg DW) of phytochemicals wherein kaempferol (1.377 μg/mg DW), apigenin (1.057 μg/mg DW), myricetin (1.022 μg/mg DW) and isorhamnetin (1.022 μg/mg DW) were recorded highly accumulated compounds in cultures at UV-C (5.4 kJ/m) dose than other UV-C radiations and light regimes.. The antioxidants activities examined as DPPH (92.8%), FRAP (182.3 µM TEAC) and ABTS (489.1 µM TEAC) were also recorded highly expressed by cultures under photoperiod after treatment with UV-C dose 5.4 kJ/m. Moreover, same cultures also expressed maximum % inhibition towards phospholipase A2 (sPLA2: 35.8%), lipoxygenase (15-LOX: 43.3%) and cyclooxygenases (COX-1: 55.3% and COX-2: 39.9%) with 1.0-, 1.3-, 1.3- and 2.8-fold increased levels as compared with control, respectively. Hence, findings suggest that light and UV can synergistically improve the metabolism of F. indica and could be used to produce such valuable metabolites on commercial scale.