Aims Cyanobacteria are immense sources of several pharmacological active compounds such as flavonoids and carotenoids with anti-inflammatory and antioxidant activity. The potential therapeutic effect of two novel cyanobacterial isolates, Cronbergia siamensis (KY296358.1) and Sphaerospermopsis aphanizomenoides (KU212886.1), against hydrogen peroxide (H2O2)-induced oxidative stress damage in the rat model was determined in this study. Methods and Results In vitro antioxidant activity of the two studied isolates was evaluated by radical scavenging assay and ferric reducing power. The possible prophylactic activity of S. aphanizomenoides (KU212886.1) against H2O2-induced oxidative stress in the rat model was assessed in vivo. Serum alanine transaminase and aspartate transaminase were measured for the liver functions in redox rats. Liver malondialdehyde (MDA), glutathione, oxidized glutathione, nitric oxide, superoxide dismutase (SOD) and catalase (CAT) were assessed as oxidative stress markers. The effect of S. aphanizomenoides on the transcripts level of superoxide dismutase (Mn-SOD) and catalase (CAT) genes in the rat's liver tissues was measured using qRT-PCR. Oral administration of S. aphanizomenoides extract in low and high doses (100, 200 mg kg(-1) b.w) resulted in significant improvement in biochemical parameters of liver functions and oxidative stress markers. Also, the endogenous antioxidant defence enzymes and the expression of their related genes (Mn/SOD, CAT) were upregulated. Immunohistochemistry of Caspase-3, an apoptotic marker, showed potent amelioration in the liver tissues. Conclusions The novel isolate S. aphanizomenoides proved in vitro and in vivo antioxidant activity against redox rat model. Significance and Impact of the Study This isolate provides a new source of pharmacological compounds with great importance in pharmacological and medical fields.