Natural antioxidant substitutes have gained popularity as alternatives to synthetic antioxidants in recent years. Microalgae are considered as an alternative natural source of antioxidants with interesting bioactive compounds. One of the fascinating bioactive features of the microalgae is their antioxidant activity. The purpose of this study was to assess the antioxidant characterizationof three microalgae: Arthrospira platensis, Scenedesmus obliquus, and Chlorella vulgaris, after treatments with gamma irradiation at a dose of 200, 300, and 700 Gy, respectively. The phytochemical examination of the three algae revealed that the maximum total antioxidant activity in S. obliquus at 300 Gy was 73.8mg g^-1 ascorbic acid, followed by A. platensis at 700 Gy by 62.9 mg g^-1 ascorbic acid. The minimum activity was noticed for C. vulgaris at 200 Gy by 46.7 mg g^-1 ascorbic acid as compared to the control. In addition, a significant increase was detected in the activity via γ-irradiation treatment of the three algae as compared to the control. Their levels were 2.26, 2.02, and 1.49mg g⁻¹FW in S. obliquus (300 Gy), A. platensis (700 Gy),and C. vulgaris (200 Gy). The antioxidant capacity of DPPH˙(2,2-diphenyl-1-picrylhydrazyl), ABTS˙⁺[2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging activity, and ferric-reducing antioxidant power (FRAP) indicated high values in S. obliquus at 300 Gy, followed by A. platensis at 700 Gy, and C. vulgaris at 200 Gy, as compared to the control. Fifteen polyphenol fractions (ten phenolics and five flavonoids) were identified, which increased significantly by gamma irradiation treatment. Therefore, treatments of A. platensis, S. obliquus, and C. vulgaris with the optimum dose of γ-irradiation increased significantly the antioxidant characterization and the content of polyphenol fractions, which directly or indirectly can help maintain the health of living beings as they are natural, safe, cheap, available, and easy to obtain.