In recent times, plants are known to be the novel sources for obtaining pharmacologically active drugs which are produced by them in response to the stress which in turn facilitate mutualistic and antagonistic interactions of the plant with its changing environment. Keeping in view the medicinal importance and vulnerability status of Bergenia ciliata in vitro cultures combined with mutagenesis prove to be better option for improvement of this plant. The reddish green callus obtained on Gamborg’s half strength medium supplemented with TDZ (2 mg/L) and NAA (2 mg/L) were irradiated with different doses of radiations and evaluated for various parameters. The maximum carotenoid content was found in 0.5 Gy treated callus while as maximum carbohydrates were present in 1.0 Gy treated callus (7.36 mg/g) as compared to non-irradiated callus (6.30 mg/g). The higher doses of gamma radiations (4.0 to 5.0 Gy) showed enhanced carbohydrate levels as compared to non-irradiated callus. The highest protein content was found in 3.0 Gy treated callus (41.1 mg/g) followed by 2.5 Gy treated callus (39.9 mg/g). The methanolic extracts of non-irradiated and irradiated callus were subjected to DPPH radical scavenging activity with maximum activity in 1.0 Gy treated sample (29.44%) followed by 1.5 Gy (23.86%). In the present study, the antioxidant enzyme activities were also evaluated (glutathione reductase, superoxide dismutase and ascorbate peroxidase) with the highest activity in 2.0 Gy treated callus as compared to non-irradiated callus. The dried callus was examined for quantification of alkaloids and tannins. Both alkaloids and tannins were significantly higher in 1.5 Gy treated callus. Gamma irradiations showed overall increase in the phenol content with highest concentration in 0.5 Gy treated callus (0.35 mg/mL). With regard to flavonoids, lower doses of gamma irradiation showed inhibitory effect on the flavonoid synthesis in the present study. The highest flavonoid content was found in 4.5 Gy (0.26 mg/mL) as compared to control (0.08 mg/mL). The maximum arbutin was found in 3.0 Gy treated sample (366.27 µg/g) while as maximum bergenin was found in non-irradiated samples (2.47 µg/g) which was analysed via HPLC.