Portulaca grandiflora belongs to the Portulacaceae family that is cultivated as an ornamental plant. Because of the sound pharmacological and curative effects of P. grandiflora, this plant can be used as a medicinal plant. Antioxidants are one of the important properties of P. grandiflora. Recurrent irradiation method using gamma-ray can modify the plant biochemical arrangement, and the resulting changes are heritable characters. The objective of the present experiment was to evaluate the phenolic content, flavonoids, and antioxidant activity of three P. grandiflora mutated genotypes in an advanced population of MV8 (eighth generation in P. grandiflora mutation breeding program). Before the extraction process, the three genotypes were measured fresh weight and dry weight. Extraction of the three test samples of P. grandiflora using the maceration method. Analysis of total phenolics was based on the Folin-Ciocalteu method and total flavonoids using the aluminum chloride solution method. The antioxidant capacity of the three P. grandiflora genotypes was examined using the free radical 1,1-diphenyl-2-picrylhydrazyl. Fresh weight and IC 50 values of the three P. grandiflora genotypes were statistically significantly different. The antioxidant activity of P. grandiflora emanated from phenolic and flavonoid compounds. The recurrent irradiation method on P. grandiflora produced three novel genotypes that were tested to have potential antioxidant capacities that were useful for protecting the body from oxidative stress.